Hafnia Phytase Variants

ABSTRACT

The present invention relates to phytases having at least 76% identity to a phytase derived from  Hafnia alvei  and comprises at least one modification in the amino acid sequence thereof. These phytase variants have modified, preferably improved, properties, such as, reduced protease sensibility, preferably they exhibit improved properties in respect of thermal performance, such as heat-stability (temperature stability, thermostability), steam stability, pelleting stability and/or temperature profile; and/or protease stability, in particular pepsin stability, pH profile, specific activity, substrate specificity, performance in animal feed (such as an improved release and/or degradation of phytate), susceptibility to glycation, and/or glycosylation pattern. The invention also relates to DNA encoding these phytases, methods of their production, as well as the use thereof, e.g., in animal feed and animal feed additives.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 12/568,312filed on Sep. 28, 2009, now allowed, which claims priority or thebenefit under 35 U.S.C. 119 of European application no. 08165245.5 filedSep. 26, 2008 and U.S. provisional application No. 61/100,784 filed Sep.29, 2008, the contents of which are fully incorporated herein byreference.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing in the form of a text file,which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a phytase which has at least 76%identity to a phytase derived from Hafnia alvei, the amino acid sequenceof which is shown in the appended sequence listing as SEQ ID NO: 2 andcomprises at least one modification as compared to this phytase (i.e.,is a variant thereof). The invention also relates to DNA encoding thesephytases, nucleic acid constructs, vectors, and host cells comprisingthe polynucleotides as well as methods of their production, as well asthe use thereof, e.g., in animal feed and animal feed additives.

BACKGROUND OF THE INVENTION Background Art

Phytases are well-known enzymes, as are the advantages of adding them tofoodstuffs for animals, including humans. Phytases have been isolatedfrom various sources, including a number of fungal and bacterialstrains.

It is an object of the present invention to provide alternativepolypeptides having phytase activity (phytases) and polynucleotidesencoding the polypeptides. The phytase variants of the invention exhibitmodified or altered preferably improved properties as compared to theparent phytase. Non-limiting examples of such properties are: Stability(such as acid-stability, heat-stability, steam stability, pelletingstability, and/or protease stability, in particular pepsin stability),temperature profile, pH profile, specific activity, substratespecificity, performance in animal feed (such as an improved releaseand/or degradation of phytate), susceptibility to glycation, and/orglycosylation pattern.

A number of three-dimensional structures of phytases of the Histidineacid phosphate (HAP) type are known. (e.g., Lim et al., 2000, NatureStruct. Biol. 7: 108-113). These phytases are structurally related, butthere are quite large differences in the amino acid sequences.

PCT/EP2008/053561 discloses the amino acid sequence of the wildtype HAPphytase of Hafnia alvei DSM 19197 (i.e., SEQ ID NO:2 herein), as SEQ IDNO:10 in PCT/EP2008/053561. The three-dimensional structure of thewildtype HAP phytase of Hafnia alvei DSM 19197 is also disclosed inPCT/EP2008/053561. The structure corresponds well with the knownstructures.

It is an object of the invention to provide phytases of modified,preferably, improved properties as compared to the parent or referencephytase from which they were derived.

Summary of Sequence Listing

In the sequence listing SEQ ID NO:1 and 2 provide DNA and amino acidsequences for the Hafnia alvei DSMZ 19197 phytase.

Summary of Examples

In the specification the following examples are provided:

Example 1: Preparation of variants, and determination of activity

Example 2: Specific activity

Example 3: Temperature stability

Example 4: Thermostability

Example 5: Temperature profile

Example 6: pH profile

Example 7: Steam Stability

Example 8: Glycation Residual activity

Example 9: Pelleting stability tests

Example 10: Performance in animal feed in an in vitro model for broilers

Example 11: Performance in an in vivo pig trial

SUMMARY OF THE INVENTION

The present invention relates to a phytase which has at least 76%identity to amino acid residues 1-413 of SEQ ID NO:2 and which comprisesat least one modification in at least one position selected from thefollowing: 139, 1, 4, 5, 6, 7, 8, 9, 10, 12, 16, 18, 25, 26, 27, 28, 29,30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 41, 45, 48, 49, 54, 55, 59, 63,64, 66, 68, 69, 70, 71, 72, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 89,91, 92, 93, 95, 96, 97, 98, 100, 101, 103, 108, 109, 110, 111, 112, 113,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 128, 130,131, 132, 133, 134, 136, 137, 138, 140, 143, 144, 145, 146, 147, 148,149, 150, 151, 152, 153, 154, 155, 156, 158, 159, 160, 161, 162, 163,168, 172, 173, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,186, 187, 189, 190, 192, 193, 194, 195, 196, 198, 199, 200, 201, 202,203, 204, 205, 206, 207, 208, 209, 211, 215, 217, 219, 221, 224, 227,228, 230, 233, 234, 235, 236, 238, 239, 240, 241, 242, 243, 244, 245,246, 247, 248, 249, 251, 256, 258, 259, 260, 261, 266, 268, 270, 279,284, 285, 286, 287, 288, 289, 290, 292, 293, 294, 295, 296, 297, 298,299, 301, 303, 304, 308, 310, 312, 313, 314, 316, 318, 319, 320, 322,324, 325, 326, 331, 335, 343, 344, 345, 346, 347, 348, 354, 355, 356,358, 360, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373,374, 375, 376, 378, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391,394, 395, 396, 397, 400, 401, 403, 404, 406, 408, 409, 411, 412, and413, wherein the positions correspond to the positions of the phytasewith the amino acids 1-413 of SEQ ID NO:2, with the proviso that thephytase is not the phytase with the amino acids 1-413 of SEQ ID NO:2.

The invention further relates to a phytase which has at least 76%identity to amino acid residues 1-413 of SEQ ID NO:2 and which comprisesat least one modification in at least one position selected from thefollowing: 139, 1, 4, 5, 6, 7, 8, 9, 10, 12, 16, 18, 25, 26, 27, 28, 29,30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 41, 45, 48, 49, 54, 55, 59, 63,64, 66, 68, 69, 70, 71, 72, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 89,91, 92, 93, 95, 96, 97, 98, 100, 101, 103, 108, 109, 110, 111, 112, 113,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 128, 130,131, 132, 133, 134, 136, 137, 138, 140, 143, 144, 145, 146, 147, 148,149, 150, 151, 152, 153, 154, 155, 156, 158, 159, 160, 161, 162, 163,168, 172, 173, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,186, 187, 189, 190, 192, 193, 194, 195, 196, 198, 199, 200, 201, 202,203, 204, 205, 206, 207, 208, 209, 211, 215, 217, 219, 221, 224, 227,228, 230, 233, 234, 235, 236, 238, 239, 240, 241, 242, 243, 244, 245,246, 247, 248, 249, 251, 256, 258, 259, 260, 261, 266, 268, 270, 279,284, 285, 286, 287, 288, 289, 290, 292, 293, 294, 295, 296, 297, 298,299, 301, 303, 304, 308, 310, 312, 313, 314, 316, 318, 319, 320, 322,324, 325, 326, 331, 335, 343, 344, 345, 346, 347, 348, 354, 355, 356,358, 360, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373,374, 375, 376, 378, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391,394, 395, 396, 397, 400, 401, 403, 404, 406, 408, 409, 411, 412, and413; and at least one further modification in at least one positionselected from the following: 1, 4, 5, 6, 7, 8, 9, 10, 12, 16, 18, 25,26, 27, 28, 29, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 41, 45, 48, 49,54, 55, 59, 63, 64, 66, 68, 69, 70, 71, 72, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 89, 91, 92, 93, 95, 96, 97, 98, 100, 101, 103, 108, 109,110, 111, 112, 113, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,125, 126, 128, 130, 131, 132, 133, 134, 136, 137, 138, 139, 140, 143,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 158,159, 160, 161, 162, 163, 168, 172, 173, 175, 176, 177, 178, 179, 180,181, 182, 183, 184, 185, 186, 187, 189, 190, 192, 193, 194, 195, 196,198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 211, 215,217, 219, 221, 224, 227, 228, 230, 233, 234, 235, 236, 238, 239, 240,241, 242, 243, 244, 245, 246, 247, 248, 249, 251, 256, 258, 259, 260,261, 266, 268, 270, 279, 284, 285, 286, 287, 288, 289, 290, 292, 293,294, 295, 296, 297, 298, 299, 301, 303, 304, 308, 310, 312, 313, 314,316, 318, 319, 320, 322, 324, 325, 326, 331, 335, 343, 344, 345, 346,347, 348, 354, 355, 356, 358, 360, 362, 363, 364, 365, 366, 367, 368,369, 370, 371, 372, 373, 374, 375, 376, 378, 382, 383, 384, 385, 386,387, 388, 389, 390, 391, 394, 395, 396, 397, 400, 401, 403, 404, 406,408, 409, 411, 412, and 413, with the proviso that the phytase is notthe phytase with the amino acids 1-413 of SEQ ID NO:2.

The invention also relates to a phytase which has at least 76% identityto amino acid residues 1-413 of SEQ ID NO:2 and which comprises at leastone modification in at least one position selected from the followingmodifications: 8L,C, 9K,P,S, 10V, 12S,R, 16V, 18K, 25A, 26R,Q, 27A, 28A,29P,R,K,A, 30P,L, 31I, 32Q,I,L, 33C,N, 35C, 36C, 37D,R,K, 38A, 41T, 45P,48H,W,N, 49L, 54C,G, 55E, 59R,K, 63C, 66C, 68L, 69E,L, 70E, 74S, 75A,76R,V, 77K,G,W, 78G,R,K,Q,S, 79L, 81E, 82S, 83G, 92Y, 93P,E,N, 95P,A,96N,V, 97R, 100W, 101C, 103A, 109D, 111R,K,S, 112S, 115L,M,RK, 116S,T,N,117A,Q, 118A,N,E,P,T, 119D,K,E, 120G,L,I,M, 121A,S,T,G,K, 122A,S,T,K,123P,M,V,A,T, 128N,R, 130L, 131R, 132T,V, 134V, 136Q, 137L, 138N,V,139C,R, 140P, 143C,V, 144R, 148R, 150C, 151D, 160G, 162N,R, 163P, 168V,172C, 173N, 175N,S176C,Q, 177C, 178C,E 179C,L,W, 181L, 185R,K, 186S,T,187E, 190N, 193R, 195L, 198E, 199C, 201C, 202A,K, 203Y, 206G,T,A,207N,L, 208A, 209S, 211T,R, 215S, 217S,G, 219M,L, 221T,G, 224C, 227Q,228C,Q, 230E, 234L,R,C,V, 235P, 236C, 239R, 243P, 244P, 245E, 246N,247D, 248T, 249S,T, 251S,D,E,R, 256D,A, 258Y, 259C, 260L, 261F,Q,A,268R, 270R, 284P, 285D, 286T, 287P, 288P, 293R,K,N, 298S, 299R, 301M,308A, 310L, 312A, 313L, 314G, 316P,A, 318E, 319L, 320N, 325C,K, 326C,331C,S,T, 335E, 343C, 344K, 346S,T, 347R, 348D,E,S,R 354L, 355S,T, 356F,358C, 360P,Q, 362M, 363C,R,K,V, 365K, 366T, 368C, 369S, 370C, 374C,P,376E, 378K, 380, 382S,T, 383N, 394N, 395E, 396D,E, 401N, 403N, and 411S.

The phytase variants of the invention exhibit modified or alteredpreferably improved properties as compared to the parent phytase.Non-limiting examples of such properties are: Stability (such asacid-stability, heat-stability, steam stability, and/or proteasestability, in particular pepsin stability), temperature profile, pHprofile, specific activity, substrate specificity, performance in animalfeed (such as an improved release and/or degradation of phytate),susceptibility to glycation, and/or glycosylation pattern. The phytasevariants of the invention preferably exhibit improved properties inrespect of thermal performance, such as heat-stability (temperaturestability, thermostability), steam stability, pelleting stability and/ortemperature profile; and/or protease stability, in particular pepsinstability, pH profile, specific activity, substrate specificity,performance in animal feed (such as an improved release and/ordegradation of phytate), susceptibility to glycation, and/orglycosylation pattern.

The invention also relates to DNA encoding these phytases, methods oftheir production, as well as the use thereof, e.g., in animal feed andanimal feed additives.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the present invention relates to a phytase which hasat least 76% identity to SEQ ID NO:2 and which comprises at least onemodification in at least one position selected from the following: 139,1, 4, 5, 6, 7, 8, 9, 10, 12, 16, 18, 25, 26, 27, 28, 29, 30, 31, 32, 33,35, 36, 37, 38, 39, 40, 41, 45, 48, 49, 54, 55, 59, 63, 64, 66, 68, 69,70, 71, 72, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 89, 91, 92, 93, 95,96, 97, 98, 100, 101, 103, 108, 109, 110, 111, 112, 113, 115, 116, 117,118, 119, 120, 121, 122, 123, 124, 125, 126, 128, 130, 131, 132, 133,134, 136, 137, 138, 140, 143, 144, 145, 146, 147, 148, 149, 150, 151,152, 153, 154, 155, 156, 158, 159, 160, 161, 162, 163, 168, 172, 173,175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 189,190, 192, 193, 194, 195, 196, 198, 199, 200, 201, 202, 203, 204, 205,206, 207, 208, 209, 211, 215, 217, 219, 221, 224, 227, 228, 230, 233,234, 235, 236, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248,249, 251, 256, 258, 259, 260, 261, 266, 268, 270, 279, 284, 285, 286,287, 288, 289, 290, 292, 293, 294, 295, 296, 297, 298, 299, 301, 303,304, 308, 310, 312, 313, 314, 316, 318, 319, 320, 322, 324, 325, 326,331, 335, 343, 344, 345, 346, 347, 348, 354, 355, 356, 358, 360, 362,363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376,378, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 394, 395, 396,397, 400, 401, 403, 404, 406, 408, 409, 411, 412, and 413. Thepercentage of identity is determined as described in the section“Phytase Polypeptides, Percentage of Identity”.

The position numbers refer to the position numbering of SEQ ID NO:2, asdescribed in the section “Position Numbering.” Positions correspondingto these SEQ ID NO:2 position numbers in other phytases are determinedas described in the section “Identifying Corresponding PositionNumbers.”

The phytase of the invention is a variant of the phytase of SEQ ID NO:2,viz. it is not identical to SEQ ID NO:2, as it comprises at least onemodification as compared to SEQ ID NO:2.

In a preferred embodiment, the phytase of the invention comprises atleast one of the following modifications: 8L,C, 9K,P,S, 10V, 12S,R, 16V,18K, 25A, 26R,Q, 27A, 28A, 29P,R,K,A, 30P,L, 31I, 32Q,I,L, 33C,N, 35C,36C, 37D,R,K, 38A, 41T, 45P, 48H,W,N, 49L, 54C,G, 55E, 59R,K, 63C, 66C,68L, 69E,L, 70E, 74S, 75A, 76R,V, 77K,G,W, 78G,R,K,Q,S, 79L, 81E, 82S,83G, 92Y, 93P,E,N, 95P,A, 96N,V, 97R, 100W, 101C, 103A, 109D, 111R,K,S,112S, 115L,M,R,K, 116S,T,N, 117A,Q, 118A,N,E,P,T, 119D,K,E, 120G,L,I,M,121A,S,T,G,K, 122A,S,T,K, 123P,M,V,A,T, 128N,R, 130L, 131R, 132T,V,134V, 136Q, 137L, 138N,V, 139C,R, 140P, 143C,V, 144R, 148R, 150C, 151D,160G, 162N,R, 163P, 168V, 172C, 173N, 175N,S 176C,Q, 177C, 178C,E179C,L,W, 181L, 185R,K, 186S,T, 187E, 190N, 193R, 195L, 198E, 199C,201C, 202A,K, 203Y, 206G,T,A, 207N,L, 208A, 209S, 211T,R, 215S, 217S,G,219M,L, 221T,G, 224C, 227Q, 228C,Q, 230E, 234L,R,C,V, 235P, 236C, 239R,243P, 244P, 245E, 246N, 247D, 248T, 249S,T, 251S,D,E,R, 256D,A, 258Y,259C, 260L, 261F,Q,A, 268R, 270R, 284P, 285D, 286T, 287P, 288P,293R,K,N, 298S, 299R, 301M, 308A, 310L, 312A, 313L, 314G, 316P,A, 318E,319L, 320N, 325C,K, 326C, 331C,S,T, 335E, 343C, 344K, 346S,T, 347R,348D,E,S,R 354L, 355S,T, 356F, 358C, 360P,Q, 362M, 363C,R,K,V, 365K,366T, 368C, 369S, 370C, 374C,P, 376E, 378K, 380, 382S,T, 383N, 394N,395E, 396D,E, 401N, 403N, and 411S.

The nomenclature used herein for modifications is described in detail inthe section “Modifications, such as Substitutions, Deletions,Insertions.”

Preferably the phytase of the invention exhibiting improvedthermostability comprises at least one of the following modifications:8C, 33C 35C, 36C, 54C, 63C, 66C, 101C, 139C, 143C, 201C, 150C, 172C,176C, 177C, 178C, 179C, 224C, 228C, 236C, 259C, 325C, 326C, 331C, 343C,358C, 363C, 368C, 370C, 374C. Specifically it comprises sets ofmodifications selected from the following: 8C/343C, 139C/201C, 179C/33C,178C/33C, 172C/35C, 177C/36C, 176C/36C, 143C/201C, 54C/101C, 63C/368C,66C/370C, 224C/236C, 150C/259C, 331C/326C, 358C/325C, 228C/363C,368C/374C. The invention also comprises such combinations as 8C/343C incombination with 139C/201C, 179C/33C, 178C/33C, 172C/35C, 177C/36C,176C/36C, 143C/201C, 54C/101C, 63C/368C, 66C/370C, 224C/236C, 150C/259C,331C/326C, 358C/325C, 228C/363C, or 368C/374C. The invention alsocomprises such combinations as 139C/201C in combination with 179C/33C,178C/33C, 172C/35C, 177C/36C, 176C/36C, 143C/201C, 54C/101C, 63C/368C,66C/370C, 224C/236C, 150C/259C, 331C/326C, 358C/325C, 228C/363C, or368C/374C. The invention also comprises such combinations as 179C/33C incombination with 178C/33C, 172C/35C, 177C/36C, 176C/36C, 143C/201C,54C/101C, 63C/368C, 66C/370C, 224C/236C, 150C/259C, 331C/326C,358C/325C, 228C/363C, or 368C/374C. The invention also comprises suchcombinations as 178C/33C in combination with 172C/35C, 177C/36C,176C/36C, 143C/201C, 54C/101C, 63C/368C, 66C/370C, 224C/236C, 150C/259C,331C/326C, 358C/325C, 228C/363C, or 368C/374C. The invention alsocomprises such combinations as 172C/35C in combination with 177C/36C,176C/36C, 143C/201C, 54C/101C, 63C/368C, 66C/370C, 224C/236C, 150C/259C,331C/326C, 358C/325C, 228C/363C, or 368C/374C. The invention alsocomprises such combinations as 177C/36C in combination with 176C/36C,143C/201C, 54C/101C, 63C/368C, 66C/370C, 224C/236C, 150C/259C,331C/326C, 358C/325C, 228C/363C, or 368C/374C. The invention alsocomprises such combinations as 176C/36C in combination with 143C/201C,54C/101C, 63C/368C, 66C/370C, 224C/236C, 150C/259C, 331C/326C,358C/325C, 228C/363C, or 368C/374C. The invention also comprises suchcombinations as 143C/201C in combination with 54C/101C, 63C/368C,66C/370C, 224C/236C, 150C/259C, 331C/326C, 358C/325C, 228C/363C, or368C/374C. The invention also comprises such combinations as 54C/101C incombination with 63C/368C, 66C/370C, 224C/236C, 150C/259C, 331C/326C,358C/325C, 228C/363C, or 368C/374C. The invention also comprises suchcombinations as 63C/368C in combination with 66C/370C, 224C/236C,150C/259C, 331C/326C, 358C/325C, 228C/363C, or 368C/374C. The inventionalso comprises such combinations as 66C/370C in combination with224C/236C, 150C/259C, 331C/326C, 358C/325C, 228C/363C, or 368C/374C. Theinvention also comprises such combinations as 224C/236C in combinationwith 150C/259C, 331C/326C, 358C/325C, 228C/363C, or 368C/374C. Theinvention also comprises such combinations as 150C/259C in combinationwith 331C/326C, 358C/325C, 228C/363C, or 368C/374C. The invention alsocomprises such combinations as 331C/326C in combination with 358C/325C,228C/363C, or 368C/374C. The invention also comprises such combinationsas 358C/325C in combination with 228C/363C, or 368C/374C. The inventionalso comprises such combinations as 228C/363C in combination with368C/3740.

In other embodiments for improving thermostability the phytase comprisesa modification selected from the following: 29P, 30P, 93P, 95P, 140P,163P, 235P, 243P, 244P, 284P, 287P, 288P, 316P, and 360P.

The phytase may also comprise a modification selected from thefollowing: 8L, 9K, 12S, 16V, 27A, 30L, 32Q, 37D, 38A, 41T, 48W, 49L,54G, 55E, 75A, 77K, 78G, 93E, 103A, 109D, 128N, 130L, 132T, 134V, 136Q,137L, 173N, 176Q, 195L, 198E, 206T, 207N, 209S, 211T, 215S, 219M, 221T,227Q, 228Q, 248T, 258Y, 260L, 261Q, 310L, 313L, 314G, 316A, 318E, 319L,320N, 335E, 354L, 356F, 360Q, 362M, 251S, 363R, 365K, 366T, 369S, 374P,376E, 378K, and 411S.

The phytase may also comprise a modification selected from thefollowing: 9R, 29R,K, 37R,K, 59R,K, 69E, 70E, 78R,K, 81E, 93E, 111R,K,115R,K, 119D, 185R,K, 230E, 239R, 245E, 251D,E, 293R,K, 348D,E, 363R,K,395E, and 396D,E.

The phytase may also comprise a modification selected from thefollowing: 12R, 25A, 26R, 28A, 29A, 30L, 45P, 48W, 76R, 97R, 117A, 118A,119D, 120L, 121A, 122A, 131R, 139R, 148R, 176R, 179L, 187E, 202A, 206A,207L, 219L, 234R, 251R, 261A, 268R, 270R, 299R, 347R, 256A, 308A, and312A.

Further the efficacy of the phytase may be improved when it comprises amodification selected from the following: 31I, 120I, I134V, N202K,D203Y, and V208A.

In specific embodiments improving the efficiency of the phytase theamino acid residues between positions 180 and 189 have been replaced bysmall peptide having a length of 4, 5, 6, 7, or 8 amino acid residues,especially the pentapeptides QADKP, GEDKP, NGISA, IAGKS, KEKHQ, KEKQQ,KEKKV, or KTDKL, and or it also comprises that the amino acid residuesbetween positions 115 and 124 have been replaced by a small peptidehaving a length of 5, 6, 7, 8, 9, 10 or 11 amino acid residues,especially the octatapeptide TQADTSSP.

In additional preferred embodiments, the phytase comprises the followingcombinations of modifications: 54C/55E/101C, 33C/178E/179C, and33C/175S/176Q/178E/179C.

The phytase of the invention may be a variant of any wildtype or variantphytase.

Specifically for the phytase of SEQ ID NO:2 the following specificmodifications are included:

M31I, 120I, I134V, N₂₀₂K, D203Y, V208A, Y179W, A221G, R321, R32L, D77G,D77W, T95A, D111S, K234C, K234V, K251S, H363V, H363R, D293R, Q93E,P348S, Q69L, Q245E, N78Q, K76V, G325K, G325G, A217G, A132T, and thefollowing combination variants:

A132V/Q162R, A132V/Q181L, A132V/E211R, A132V/D83G, A132V/A217G,A132V/A217S, E100W/H363R D138V/Y48H, A132V/A217G,A132V/Q162R/Q181L/A217G, P348R/H363R, Q9S/D92Y, Q9P/L10V/D92Y/H115M,Q9P/L10V/D92Y/H115/L, D92Y/H115M, D92Y/H115M/L, D92Y/H115M,E100W/A217G/H363R, A217G/K251S, E100W/A217G/K251S, E100W/K251S,E100W/1555V/A217G, Q9S/E100W/R160G/A217G/H363R, D92Y/E100W/A217G/H363R,E100W/H115M/A217G/H363R, E100W/A217G/P348R/H363R,Q9S/A89A/D92Y/H115M/A217G/H363R, N78Q/E100W/A217G/H363R,K76V/N78Q/E100W/A217G/H363R, D83G/E100W/A217G/H363R,E100W/Y179W/A217G/H363R, E100W/A217G/K234V/K251E/1286T/H363R,E100W/A217G/K234V/P348R/H363R,Q9S/R18K/A89A/D92Y/H115M/A217G/K234V/H363R,Q9S/D92Y/H115M/A217G/K234V/H363R,Q9S/N78Q/D92Y/L112S/H115M/K234V/P348R/H363R,Q9S/N78Q/A89A/D92Y/H115M/A132V/Q162R/Q181L/A217G/K234V/P348R,Q9S/E54C/D92Y/A101C/H143C/Q193R/I201C/A217G/H363R,E54C/N78S/D92Y/A101C/H143C/L199C/A217G/H363R,E54C/A101C/M168V/A217G/H363R, P82S/D92Y/E100W/H143C/I201C/A217G/H363R,P82S/D92Y/E100W/H143C/I201C/A217G/H363R,Q9S/N78Q/D92Y/L112S/H115M/A217G/K234V/P348R/H363R,D92Y/A217G/K234V/H363R, Y64S/D92Y/E100WN179W/A217G/H363R,D92Y/A217G/H363R,Q9S/N78Q/A89A/D92Y/H115M/A132V/H1430/Q162R/Q181L/I201C/A217G/K234V/P348R,Q9S/N78Q/A89A/D92Y/H115M/A132V/K139C/Q162R/Q181L/I201C/A217G/K234V/P348R,Q9S/N78Q/A89A/D92Y/H115M/A132V/K139C/Q162R/Q181L/L1990/A217G/K234V/L301M/P348R, Q9S/N78Q/A89A/D92Y/H115M/A132V/Q162RN179W/Q181L/A217G/K234V/P348R,D92Y/E100W/A217G/H363R/+D33CN179C,D92Y/E100W/A217G/H363R/+116−123(HQQNTQQA->TQADTSSP)=D92Y/E100W/H116T/Q118A/N119D/Q121S/Q122S/A123P/A217G/H363R,Q9S/E54C/D92Y/A101C/H143C/Q193R/I201C/A217G/N298S/H363R+116−123(HQQNTQQA->TQADTSSP)=Q9S/E54C/D92Y/A101C/H116T/Q118A/N119D/Q121S/Q122S/A123P/H143C/Q193R/I201C/A217G/N298S/H363R,Q9S/N78Q/A89A/D92Y/H115M/A132V/Q162RN179W/A217G/K234V/P348R/H363R,Q9S/N78Q/A89A/D92Y/H115M/A132V/Q162R/Y179W/A217G/K234V/S261F/P348R/H363R,Q9S/N78Q/A89A/D92Y/H115M/A132V/K139C/G151D/Q162RN179W/Q181L/I201C/A217G/K234V/P348R,D92Y/E100W/K139C/I201C/A217G/N247D/H363R,E54C/D92Y/A101C/M168V/A217G/H363R,Q9S/N78Q/A132V/K139C/Q162RN179W/I201C/A217G/K234L/P348R/H363R,D92Y/E100W/H1430/A144R/I201C/A217G/N247D/H363R,D92Y/E100W/H116S/K139C/I201C/A217G/N247D/H363R,D92Y/E100W/H128R/K139C/H143V/I201C/A217G/N247D/H363R,D92Y/E100W/K139C/I201C/N₂₀₆G/A217G/N247D/H363R,D92Y/E100W/K139C/I201C/A217G/N247D/H363R,D92Y/E100W/K139C/I201C/A217G/N247D/Q256D/H363R,D92Y/E100W/K139C/I201C/A217G/N247D/H363R,D92Y/E100W/K139C/I201C/A217G/N247D/N344K/H363R,D92Y/E100W/K139C/A144S/K176E/I201C/A217G/K234V/N247D/H363R,D92Y/E100W/K139C/I201C/A217G/K234V/N247D/H363R/E54C/H55E/A1010,D92Y/E100W/K139C/T152A/I201C/A217G/K234V/N247D/H363R,Y48H/D92Y/E100W/K139C/T152C/I201C/A217G/K234V/N247D/H363R,D92Y/E100W/K139C/I201C/A217G/K234V/N247D/S2840/H363R,D92Y/E100W/K139C/I201C/A217G/K234V/N247D/T287W/H363R,D92Y/E100W/K139C/I201C/A217G/K234V/N247D/R289M/H363R,Y48H/D92Y/E100W/K139C/I201C/A217G/K234V/N247D/R289W/H363R,N78Q/D92Y/E100W/K139C/I201C/A217G/K234V/N247D/Q256D/H363R,D92Y/E100W/K139C/I201C/A217G/K234V/N247D/Q256D/P348R/H363R,D92Y/E100W/K139C/Q162R/Q181L/I201C/A217G/K234V/N247D/Q256D/H363R,D92Y/E100W/A113G/K139C/I201C/A217G/K234V/N247D/H363R, D92Y/E100W/T120Gor A*/K139C/I201C/A217G/K234V/N247D/H363R/L395L or V,D92Y/E100W/K139C/I201C/A217G/K234V/N247D/S284M/H363R,D92Y/E100W/K139C/I201C/A217G/K234V/N247D/H363R/A366S,D92Y/E100W/K139C/I201C/A217G/K234V/N247W/Q256D/H363R,D92Y/E100W/H128R/K139C/I201C/A217G/K234V/N247E/Q256D/H363R,D92Y/E100W/K139C/Q141S/I201C/A217G/K234V/N247D/H363R,D92Y/E100W/K139C/A144S/I201C/A217G/K234V/N247D/H363R,P75N/K76N/D77Q/N78T/D92Y/E100W/K139C/I201C/A217G/K234V/N247D/Q256D/H363R,D92Y/E100W/K139C/D173N/P175S/I201C/A217G/K234V/N247D/Q256D/H363R,D92Y/E100W/K139C/T152I/I201C/A217G/K234V/N247D/Q256D/1294T/H363R, D33N/D92Y/E100W/K139C/I201C/A217G/K234V/N247 D/Q256D/H363R,Y48H/D92Y/E100W/K139C/T152C/I201C/A217G/K234V/N247D/Q256D/H363R,D92Y/E100W/K139C/T152A/I201C/A217G/K234V/N247D/Q256D/H363R,D92Y/T98S/E100W/K139C/T152A/I201C/A217G/K234V/N247D/Q256D/H363R,D92Y/T98S/E100W/K139C/T152A/L199S/I201C/A217G/K234V/N247W/Q256D/H363R,Y48H/D92Y/T98S/E100W/K139C/T152I/I201C/A217G/K234V/N247W/Q256D/H363R,E54C/D92Y/A101C/K139C/I201C/A217G/K234V/N247D/Q256D/H363R,Y48H/E54C/D92Y/A101C/K139C/I201C/A217G/K234V/N247D/R289W/H363R,D92Y/T98S/E100W/K139C/T152A/I201C/A217G/K234V/N247W/Q256D/R289W/H363R,Y48H/D92/E100W/K139C/T152C/I201C/A217G/K234V/N247W/Q256D/R289W/H363R,Y48H/D92Y/E100W/K139C/I201C/A217G/K234V/N247W/Q256D/H363R,Y48H/D92Y/E100W/K139C/T152A/I201C/A217G/K234V/N247D/R289W/H363R,Y48H/D92Y/E100W/K139C/T152A/I201C/A217G/K234V/N247W/Q256D/H363R,Y48H/E54C/D92Y/A101C/K139C/T152A/I201C/A217G/K234V/N247D/R289W/H363R,Y48H/E54C/D92Y/A101C/K139C/I201C/A217G/K234V/N247W/R289W/H363R,Y48H/E54C/D92Y/A101C/K139C/T152I201C/A217G/K234V/N247W/R289W/H363R,Y48H/E54C/D92Y/E100W/A101C/K139C/T152A/I201C/A217G/K234V/N247W/Q256D/H363R,Y48H/E54C/D92Y/A101C/K139C/T152A/I201C/V₂₀₈T/A217G/K234V/N247D/R289W/H363R,T35A/Y48H/E54C/P75N/K76N/D77Q/N78T/D92Y/A101C/K139C/T152A/I201C/A217G/K234V/N247D/R289W/H363R,Y48H/E54C/D92Y/A101C/K139C/T152A/I201C/K207Q/V208T/A217G/K234V/N247D/R289W/H363R,E540/D92Y/A101C/K139C/I201C/A217G/Q256D/H363R,E54C/P75N/K76N/D77Q/N78T/D92Y/A101C/K139C/I201C/A217G/H363R,E540/D92Y/A101C/K139C/I201C/V208T/A217G/H363R,E54C/P75N/K76N/D77Q/N78T/D92Y/A101C/K139C/I201C/V208T/A217G/H363R,Y48H/E54C/P75N/K76N/D77Q/N78T/D92Y/A101C/K139C/T152A/I201C/V₂₀₈T/A217G/K234V/N247D/R289W/H363R,E54C/P75N/K76N/D77Q/N78T/D92Y/A101C/K139C/I201C/V208T/A217G/K234V/N239S/N247D/Q256D/H363R.

The invention also relates to a method for producing a phytase variant,of a reference or parent phytase having at least 76% identity to SEQ IDNO:2 whereby said variant exhibits at least one substitution, insertionor deletion in one or more of the positions: 139, 1, 4, 5, 6, 7, 8, 9,10, 12, 16, 18, 25, 26, 27, 28, 29, 30, 31, 32, 33, 35, 36, 37, 38, 39,40, 41, 45, 48, 49, 54, 55, 59, 63, 64, 66, 68, 69, 70, 71, 72, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 89, 91, 92, 93, 95, 96, 97, 98, 100,101, 103, 108, 109, 110, 111, 112, 113, 115, 116, 117, 118, 119, 120,121, 122, 123, 124, 125, 126, 128, 130, 131, 132, 133, 134, 136, 137,138, 140, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 158, 159, 160, 161, 162, 163, 168, 172, 173, 175, 176, 177,178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 189, 190, 192, 193,194, 195, 196, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,209, 211, 215, 217, 219, 221, 224, 227, 228, 230, 233, 234, 235, 236,238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 251, 256,258, 259, 260, 261, 266, 268, 270, 279, 284, 285, 286, 287, 288, 289,290, 292, 293, 294, 295, 296, 297, 298, 299, 301, 303, 304, 308, 310,312, 313, 314, 316, 318, 319, 320, 322, 324, 325, 326, 331, 335, 343,344, 345, 346, 347, 348, 354, 355, 356, 358, 360, 362, 363, 364, 365,366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 378, 382, 383,384, 385, 386, 387, 388, 389, 390, 391, 394, 395, 396, 397, 400, 401,403, 404, 406, 408, 409, 411, 412, and 413, wherein the positionscorrespond to the positions of the phytase with the amino acids 1-413 ofSEQ ID NO:2, the method comprising, with the proviso that the variant isnot the phytase with the amino acids 1-413 of SEQ ID NO:2,

a) mutating the DNA or gene encoding the parent phytase in a mannerwhereby the DNA or gene encodes for said substitution, insertion and/ordeletion,

b) operably linking said DNA or gene to one or more control sequencesthat direct the production of the phytase in a suitable expression hostto create a DNA construct or a recombinant expression vector,

c) transferring said construct or vector to a suitable host,

d) cultivating said host to produce the variant phytase, and e)recovering the phytase,

Specifically the method provides for variants having improved propertiesin respect of thermal performance, including heat-stability, temperaturestability, thermostability, steam stability, pelleting stability, and/ortemperature profile, and/or an improved efficiency, including animproved pH profile, an improved specific activity, an alteredglycosylation pattern, an improved performance in animal feed, and/orwhich incorporates a change of a potential protease cleavage site and/orglycation site.

Strategy for Preparing Variants

The structure of the H. alvei DSM 19197 phytase (amino acids 1 to 413 ofSEQ ID NO:2) is disclosed in PCT/EP200C/053561.

The structure was subjected to molecular dynamics (MD) simulations andelectrostatic calculations. Positions for putative disulfide bridges andprolines were also identified, as well as other positions of potentialimportance as regards the various desirable enzymatic properties.Finally, putative glycosylation sites (stretches of NXT or NXS) wereidentified.

All these suggestions were evaluated within the framework of themodelled structure and the simulation results, for the thermostabilityproperty with particular emphasis at the high temperature end.

The corresponding phytase variants were prepared by methods known in theart and tested as described in the experimental part.

Phytase Polypeptides, Percentage of Identity

In the present context a phytase is a polypeptide having phytaseactivity, i.e., an enzyme which catalyzes the hydrolysis of phytate(myo-inositol hexakisphosphate) to (1) myo-inositol and/or (2) mono-,di-, tri-, tetra- and/or penta-phosphates thereof and (3) inorganicphosphate.

In the present context the term a phytase substrate encompasses, i.a.,phytic acid and any phytate (salt of phytic acid), as well as thephosphates listed under (2) above.

The ENZYME site at the internet (www.expasy.ch/enzyme/) is a repositoryof information relative to the nomenclature of enzymes. It is primarilybased on the recommendations of the Nomenclature Committee of theInternational Union of Biochemistry and Molecular Biology (IUB-MB) andit describes each type of characterized enzyme for which an EC (EnzymeCommission) number has been provided (Bairoch A. The ENZYME database,2000, Nucleic Acids Res. 28: 304-305). See also the handbook EnzymeNomenclature from NC-IUBMB, 1992).

According to the ENZYME site, three different types of phytases areknown: A so-called 3-phytase (alternative name 1-phytase; a myo-inositolhexaphosphate 3-phosphohydrolase, EC 3.1.3.8), a so-called 4-phytase(alternative name 6-phytase, name based on 1L-numbering system and not1D-numbering, EC 3.1.3.26), and a so-called 5-phytase (EC 3.1.3.72). Forthe purposes of the present invention, all three types are included inthe definition of phytase.

In a particular embodiment, the phytases of the invention belong to thefamily of histidine acid phosphatases (HAP), which includes theEscherichia coli pH 2.5 acid phosphatase (gene appA), as well as fungalphytases such as Aspergillus awamorii phytases A and B (EC: 3.1.3.8)(gene phyA and phyB). The histidine acid phosphatases share two regionsof sequence similarity, each centered around a conserved histidineresidue. These two histidines seem to be involved in the enzymes'catalytic mechanism. The first histidine is located in the N-terminalsection and forms a phosphor-histidine intermediate while the second islocated in the C-terminal section and possibly acts as proton donor.

In a further particular embodiment, the phytases of the invention have aconserved active site motif, viz. R—H-G-X—R—X—P, wherein X designatesany amino acid (see amino acids 18 to 24 of SEQ ID NO:2). In a preferredembodiment, the conserved active site motif is R—H-G-V—R-A-P, i.e.,amino acids 18-24 (by reference to SEQ ID NO:2) are RHGVRAP.

For the purposes of the present invention the phytase activity isdetermined in the unit of FYT, one FYT being the amount of enzyme thatliberates 1 micro-mol inorganic ortho-phosphate per min. under thefollowing conditions: pH 5.5; temperature 37° C.; substrate: sodiumphytate (C₆H₆O₂₄P₆Na₁₂) in a concentration of 0.0050 mol/l. Suitablephytase assays are the FYT and FTU assays described in Example 1 of WO00/20569. FTU is for determining phytase activity in feed and premix.Phytase activity may also be determined using the assays of Example 1(“Determination of phosphatase activity” or “Determination of phytaseactivity”).

In a particular embodiment the phytase of the invention is isolated. Theterm “isolated” as used herein refers to a polypeptide which is at least20% pure, preferably at least 40% pure, more preferably at least 60%pure, even more preferably at least 80% pure, most preferably at least90% pure, and even most preferably at least 95% pure, as determined bySDS-PAGE. In particular, it is preferred that the polypeptides are in“essentially pure form”, i.e., that the polypeptide preparation isessentially free of other polypeptide material with which it is nativelyassociated. This can be accomplished, for example, by preparing thepolypeptide by means of well-known recombinant methods or by classicalpurification methods.

The relatedness between two amino acid sequences is described by theparameter “identity”. For purposes of the present invention, thealignment of two amino acid sequences is determined by using the Needleprogram from the EMBOSS package (http://emboss.org) version 2.8.0. TheNeedle program implements the global alignment algorithm described inNeedleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453. The substitutionmatrix used is BLOSUM62, gap opening penalty is 10, and gap extensionpenalty is 0.5.

The degree of identity between an amino acid sequence of the presentinvention (“invention sequence”) and the amino acid sequence referred toin the claims (SEQ ID NO:2) is calculated as the number of exact matchesin an alignment of the two sequences, divided by the length of the“invention sequence,” or the length of the SEQ ID NO:2, whichever is theshortest. The result is expressed in percent identity.

An exact match occurs when the “invention sequence” and SEQ ID NO:2 haveidentical amino acid residues in the same positions of the overlap (inthe alignment example below this is represented by “|”). The length of asequence is the number of amino acid residues in the sequence (e.g., thelength of amino acids 1-413 of SEQ ID NO:2 is 413).

For further detailed explanation reference is made to WO 2007/112739 atpage 7, line 24 to page 8, line 5.

In particular embodiments of the phytase of the invention, the degree ofidentity to SEQ ID NO:2 is at least 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or at least 99%. In still further particular embodiments, thedegree of identity is at least 98.0%, 98.2%, 98.4%, 98.6%, 98.8%, 99.0%,99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or at least99.9%. In alternative embodiments, the degree of identity is at least70%, 71%, 72%, or at least 73%.

In still further particular embodiments, the phytase of the inventionhas no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or no more than 10modifications as compared to SEQ ID NO:2; no more than 11, 12, 13, 14,15, 16, 17, 18, 19, or no more than 20 modifications as compared to SEQID NO:2; no more than 21, 22, 23, 24, 25, 26, 27, 28, 29, or no morethan 30 modifications as compared to SEQ ID NO:2; no more than 31, 32,33, 34, 35, 36, 37, 38, 39, or not more than 40 modifications ascompared to SEQ ID NO:2; no more than 41, 42, 43, 44, 45, 46, 47, 48,49, or no more than 50 modifications as compared to SEQ ID NO:2; no morethan 51, 52, 53, 54, 55, 56, 57, 58, 59, or no more than 60modifications as compared to SEQ ID NO:2; no more than 61, 62, 63, 64,65, 66, 67, 68, 69, or no more than 70 modifications as compared to SEQID NO:2; no more than 71, 72, 73, 74, 75, 76, 77, 78, 79, or no morethan 80 modifications as compared to SEQ ID NO:2; no more than 81, 82,83, 84, 85, 86, 87, 88, 89, or no more than 90 modifications as comparedto SEQ ID NO:2; no more than 91, 92, 93, 94, 95, 96, 97, 98, 99, or nomore than 100 modifications as compared to SEQ ID NO:2; no more than101, 102, 103, 104, 105, 106, 107, 108, 109, or no more than 110modifications as compared to SEQ ID NO:2.

Position Numbering

The nomenclature used herein for defining amino acid positions is basedon the amino acid sequence of the phytase derived from Hafnia alvei DSM19197, the mature sequence of which is given in the sequence listing asSEQ ID NO:2 (amino acids 1-413 of SEQ ID NO:2). Accordingly, in thepresent context, the basis for numbering positions is SEQ ID NO:2starting with S1 and ending with P413.

When used herein the term “mature” part (or sequence) refers to thatpart of the polypeptide which is secreted by a cell which contains, aspart of its genetic equipment, a polynucleotide encoding thepolypeptide. In other words, the mature polypeptide part refers to thatpart of the polypeptide which remains after the signal peptide part, aswell as a propeptide part, if any, has been cleaved off. The signalpeptide part can be predicted by programs known in the art (e.g.,SignalP). SEQ ID NO:2 is the expected mature part. Generally, the firstamino acid of the mature part of an enzyme can be determined byN-terminal sequencing of the purified enzyme. Any difference between thesignal peptide part and the mature part must then be due to the presenceof a propeptide.

Modifications, Such as Substitutions, Deletions, Insertions

A phytase variant can comprise various types of modifications relativeto a template (i.e., a parent or reference phytase, or a comparativeamino acid sequence such as SEQ ID NO:2): An amino acid can besubstituted with another amino acid; an amino acid can be deleted; anamino acid can be inserted between two residues; as well as anycombination of any number of such modifications. In the present contextthe term “insertion” is intended to cover also N- and/or C-terminalextensions.

The general nomenclature used herein for a single modification is thefollowing: XDcY, where “X” and “Y” independently designate a one-letteramino acid code, or a “*” (deletion of an amino acid), “D” designates anumber, and “c” designates an alphabetical counter (a, b, c, and soforth), which is only present in insertions. Reference is made to Table1 below which describes purely hypothetical examples of applying thisnomenclature to various types of modifications.

TABLE 1 Nomenclature examples Type Description Example Sub- stitu- tionX = Amino  acid in  template D = Position in template c empty Y = Amino acid in  variant G80A  

In- ser- tion X = “*” D = Position in template before the  insertion c =“a” for  first  insertion  at this  position,  “b” for  next, etc*80aT *80bY *85aS  

Dele- tion X = Amino  acid in  template D = Position  in templatec empty Y = “*” V81*  

N-ter- minal exten- sion Insertions  at position  “0”. *0aA *0bT *0cG  

C-ter- minal exten- sion Insertions  after the  N-terminal  amino acid.*275aS *275bT  

As explained above, the position number (“D”) is counted from the firstamino acid residue of SEQ ID NO:2.

Several modifications in the same sequence are separated by “I” (slash),e.g., the designation “1*/2*/3*” means that the amino acids in positionnumber 1, 2, and 3 are all deleted, and the designation “104A/105F”means that the amino acid in position number 104 is substituted by A,and the amino acid in position number 105 is substituted by F.

Alternative modifications are separated by “,” (comma), e.g., thedesignation “119R,K” means that the amino acid in position 119 issubstituted with R or K.

The commas used herein in various other enumerations of possibilitiesmean what they usually do grammatically, viz. often and/or. E.g., thefirst comma in the listing “53V,Q, 121D, and/or 167Q” denotes analternative (V or Q), whereas the two next commas should be interpretedas and/or options: 53V or 53Q, and/or 121D, and/or 167Q.

In the present context, “at least one” (e.g., modification) means one ormore, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modifications; or 12, 14,15, 16, 18, 20, 22, 24, 25, 28, or 30 modifications; and so on, up to amaximum number of modifications of 125, 130, 140, 150, 160, 170, 180,190, or of 200. The phytase variants of the invention, however, stillhave to be at least 76% identical to SEQ ID NO:2, this percentage beingdetermined as described above.

A substitution or extension without any indication of what to substituteor extend with refers to the insertion of any natural, or non-natural,amino acid, except the one that occupies this position in the template.

Identifying Corresponding Position Numbers

As explained above, the mature phytase of Hafnia alvei DSM 19197 (SEQ IDNO:2) is used as the standard for position numbering and, thereby, alsofor the nomenclature.

For another phytase, in particular a phytase variant of the invention,the position corresponding to position D in SEQ ID NO:2 is found byaligning the two sequences as specified above in the section entitled“Phytase polypeptides, percentage of identity”. From the alignment, theposition in the sequence of the invention corresponding to position D ofSEQ ID NO:2 can be clearly and unambiguously identified (the twopositions on top of each other in the alignment).

Below some additional, purely hypothetical, examples are included whichare derived from Table 1 above which in the third column includes anumber of alignments of two sequences:

Consider the third cell in the first row of Table 1: The upper sequenceis the template, the lower the variant. Position number 80 refers toamino acid residue G in the template. Amino acid A occupies thecorresponding position in the variant. Accordingly, this substitution isdesignated G80A.

Consider now the third cell in the second row of Table 1: The uppersequence is again the template and the lower the variant. Positionnumber 80 again refers to amino acid residue G in the template. Thevariant has two insertions, viz. TY, after G80 and before V81 in thetemplate. Whereas the T and Y of course would have their own “real”position number in the variant amino acid sequence, for the presentpurposes we always refer to the template position numbers, andaccordingly the T and the Y are said to be in position number 80a and80b, respectively.

Finally, consider the third cell in the last row of Table 1: Positionnumber 275 refers to the last amino acid of the template. A C-terminalextension of ST are said to be in position number 275a and 275b,respectively, although, again, of course they have their own “real”position number in the variant amino acid sequence.

Modified Properties, Reference or Parent Phytase

In a particular embodiment, the phytase of the invention has altered ormodified, preferably improved, properties. The terms “altered”,“modified” and “improved” imply a comparison with another phytase.Examples of such other, reference, parent or comparative, phytases are:SEQ ID NO:2, and/or other phytases having a sequence identity to SEQ IDNO:2 of more than 76%, preferably more than 80, 85, 90, 95, or 98%

Non-limiting examples of properties that are modified, preferablyimproved, are the following: Thermostability, steam stability, pelletingstability, pH profile, specific activity, performance in animal feed,protease-sensibility, and/or glycosylation pattern. The phytase of theinvention may also have an altered, preferably improved, temperatureprofile, and/or it may incorporate changes of a potential proteasecleavage sites to reduce the protease sensibility. Especially thethermal performance, including heat-stability, temperature stability,thermostability, steam stability, and/or pelleting stability isconsidered an important characteristic or proproperty,

Thermal Performance Temperature-Stability

Temperature stability may be determined as described in Example 3 bydetermining the residual activity after incubation for 30 minutes attemperatures from 70° C. to 80° C.

Thermostability

Thermostability may be determined as described in Example 4, i.e., usingDSC measurements to determine the denaturation temperature, Td, of thepurified phytase protein. The Td is indicative of the thermostability ofthe protein: The higher the Td, the higher the thermostability.Accordingly, in a preferred embodiment, the phytase of the invention hasa Td which is higher than the Td of a reference phytase, wherein Td isdetermined on purified phytase samples (preferably with a purity of atleast 90% or 95%, determined by SDS-PAGE).

Heat-Stability

Heat stability may be determined as described in Example 5 bydetermining the temperature/activity profile of the variant phytases.

Steam Stability

Steam stability may be determined as described in Example 7 bydetermining the residual activity of phytase molecules after steamtreatment at 85° C. or 90° C. for a short time.

Pelleting Stability

Pelleting stability may be determined as described in Example 9 by usingenzyme granulate pre-mixed with feed. This premix is mixed with feed.From the mixer the feed is conditioned with steam to 95° C. Afterconditioning the feed is pressed to pellets and the residual activitydetermined.

In preferred embodiments, the thermal properties such as heat-stability,temperature stability, thermostability, steam stability, and/orpelleting stability as provided by the residual activity, Td or otherparameter of the phytase of the invention is higher than thecorresponding value, such as the residual activity or Td, of the phytaseof SEQ ID NO:2, more preferably at least 101% thereof, or at least 102%,103%, 104%, 105%, 106%, 107%, 108%, 109%, or at least 110% thereof. Evenmore preferably, the value of the parameter, such as residual activityor Td, of the phytase of the invention is at least 120%, 130%, 140%,150%, 160%, 170%, 180%, or at least 190% of the value for the phytase ofSEQ ID NO:2.

In still further particular embodiments, the thermostable phytase of theinvention has a melting temperature, Tm (or a denaturation temperature,Td), as determined using Differential Scanning calorimetry (DSC) asdescribed in the Examples (i.e., in 20 mM sodium acetate, pH 4.0), of atleast 50° C. In still further particular embodiments, the Tm is at least51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 62.5. 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or at least100° C. DSC measurements may also be performed as described in theExamples.

The structure disclosed in PCT/EP2008/053561 was used to identifypositions that are selected for modification. The structure was alsocompared to other known HAP phytase structures for the same purpose.

Using Molecular Dynamics simulations to analyse mobilities at hightemperatures the following positions were identified for modification toprovide improved thermal properties: 4, 5, 6, 7, 8, 9, 26, 27, 28, 29,30, 32, 33, 37, 38, 39, 40, 41, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 91, 108, 109, 110, 111, 117, 118, 119, 120, 121,122, 131, 132, 133, 134, 138, 139, 140, 144, 145, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 158, 159, 160, 161, 163, 175, 178, 179,180, 181, 182, 183, 184, 185, 186, 187, 189, 190, 193, 194, 196, 198,200, 201, 202, 203, 204, 205, 206, 207, 233, 234, 235, 236, 238, 239,240, 241, 242, 243, 244, 285, 286, 287, 288, 289, 290, 292, 293, 294,295, 296, 297, 319, 320, 322, 324, 343, 344, 345, 346, 347, 364, 365,366, 367, 369, 370, 371, 372, 373, 375, 382, 383, 384, 385, 386, 387,388, 389, 390, 391, 396, 397, 400, 403, 404, 408, 409, 412, and 413.

It is specifically proposed that the modifications in some of thesepositions are selected from the following: 8L,C,M,Y, 9K,P,S, 26R,Q,H,27A, 28A, 29P,R,K,A, 30P,L, 32Q,I,L, 33C,N, 37D,R,K, 38A, 41T,Q, 69E,L,70E, 72A, 74S, 75A, 76R,V,N, 77K,G,W,Q, 78G,R,K,Q,S,T, 79L, 81E, 82S,83G, 109D,G, 111R,K,S, 117A,Q, 118A,N,E,P,T, 119D,K,E, 120G,L,I,M,121A,S,T,G,K, 122A,S,T,K, 131R,Q, 132T,V, 134V, 138N,V, 139C,R, 140P,144R, 148R, 150C, 151D, 152A,G,T,I, 160G, 163P,K, 175N,S 178C,E179C,L,W, 181L, 185R,K, 186S,T, 187E, 190N, 193R, 198E, 201C,G,V,202A,K, 203Y, 206G,T,A, 207N,L,Q,R, 234L,R,C,V,E, 235P, 236C, 239R,K,242S, 243P, 244P, 285D, 286T, 287P, 288P, 289W, 293R,K,N, 294L, 319L,320N, 343C, 344K, 346S,T, 347R, 365K, 366T, 369S,D, 370C, 382S,T, 383N,396D,E,K 403N, and 411S.

Based on comparisons to other known phytases the following positionswere identitied to be able to provide improved thermal properties, suchas thermostability: 12, 16, 48, 49, 54, 55, 77, 93, 100, 103, 128, 130,136, 137, 173, 176, 195, 209, 211, 215, 219, 221, 227, 228, 248, 251,258, 260, 261, 310, 313, 314, 316, 318, 335, 354, 356, 360, 362, 363,374, 376, 378, and 411.

From the comparisons it is specifically indicated that the substitutionsshould be chosen among the following 12S, 16V, 48W, 49L, 54G, 55E, 77K,93E, 100W, 103A, 128N, 130L, 136Q, 137L, 173N, 176Q, 195L, 209S, 211T,215S, 219M, 221T, 227Q, 228Q, 248T, 251S, 258Y, 260L, 261Q, 310L, 313L,314G, 316A, 318E, 335E, 354L, 356F, 360Q, 362M, 363R, 374P, 376E, 378K,and 411S

In relation to variants produced from the phytase of SEQ ID NO:2 themodifications should be chosen from the following: K12S, L16V, Y48W,I49L, E54G, H55E, D77K, Q93E, L103A, H128N, V130L, S136Q, M137L, D173N,K176Q, M195L, A209S, E211T, G2155, T219M, A221T, E227Q, H228Q, S248T,K251S, D258Y, M260L, S261Q, I310L, I313L, S314G, M316A, G318E, A335E,M354L, Y356F, A360Q, L362M, H363R, A374P, S376E, R378K, and/or Q411S.

In particular embodiments, whereby disulfide bridges are created in themolecule, an improved thermostability is expected from the followingvariants of a phytase having at least 76% identity to amino acidresidues 2-413 of SEQ ID NO:2: 8C/343C, 139C/201C, 179C/33C, 178C/33C,172C/35C, 177C/36C, 176C/36C, 143C/201C, 54C/101C, 63C/368C, 66C/370C,224C/236C, 150C/259C, 331C/326C, 358C/325C, 228C/363C, and 368C/374C.

Similarly an improved thermostability is also expected from substitutingproline residues for the existing residues in selected positions. Thisis expected from the following phytase variants: 29P, 30P, 93P, 95P,140P, 163P, 235P, 243P, 244P, 284P, 287P, 288P, 316P, and 360P.Specifically for modifications in SEQ ID NO:2 the followingmodifications should improve thermal stability Q29P, T30P, Q93P, K95P,S140P, A163P, V235P, E243P, Q244P, S284P, T287P, S288P, M316P, andA360P.

Also, the optimization of charged residues is able to improve thermalproperties, such as thermostability. The optimization relates to thecharge-charge interactions on the surface of the phytase molecule.

Three groups of substitutions are listed below for modifying parent orreference phytases having at least 76% identity to amino acid residues1-413 of SEQ ID NO:2 with residues as indicated. The residues whosecharge may be inverted, residues changed to a negative charge, andresidues to be changed to a positive charge are:

Charge Inversion

D111R,K, K251D,E and D293R,K.

Change to Negative

Q69E, Q70E, T81E, Q93E, N119D, Q230E, Q245E, P348D,E, L395E, andS396D,E.

Change to Positive

Q9R, Q29R,K, H37R,K, L59R,K, N78R,K, H115R,K, 1185R,K, N239R andH363R,K.

Temperature Profile/Temperature Stability

Whether or not a phytase of the invention has a modified temperatureprofile as compared to a reference phytase may be determined asdescribed in Example 5. Accordingly, in a particular embodiment thephytase of the invention has a modified temperature profile as comparedto a reference phytase, wherein the temperature profile is determined asphytase activity as a function of temperature on sodium phytate at pH5.5 in the temperature range of 20-90° C. (in 10° C. steps). A preferredbuffer is in 0.25 M Na-acetate buffer pH 5.5. The activity at eachtemperature is preferably indicated as relative activity (in %)normalized to the value at optimum temperature. The optimum temperatureis that temperature within the tested temperatures (i.e., those with5-10° C. jumps) where the activity is highest.

pH Profile

Whether or not a phytase of the invention has an altered pH profile ascompared to a reference phytase may be determined as described in theExamples. Accordingly, in a particular embodiment the phytase of theinvention has an altered pH profile as compared to a reference phytase,wherein the pH profile is determined as phytase activity as a functionof pH on sodium phytate at 37° C. in the pH range of 2.0 to 7.5 (in 0.5pH-unit steps). A preferred buffer is a cocktail of 50 mM glycine, 50 mMacetic acid and 50 mM Bis-Tris. The activity at each pH is preferablyindicated as relative activity (in %) normalized to the value at optimumpH.

An example of an altered pH profile is where the pH curve (relativeactivity as a function of pH) is shifted towards higher, or lower, pH.Preferred substitutions which provide a shift of 0.5 pH units towards ahigher pH as compared to the reference phytase of SEQ ID NO:2. However,for certain purposes it may be preferred to provide a shift of 0.5 pHunits towards a lower pH as compared to the reference phytase of SEQ IDNO:2.

Another example of an altered pH profile is where the optimum pH ischanged, in the upward or the downward direction.

In a particular embodiment, the phytase of the invention has an alteredpH profile as compared to a reference phytase. More in particular, thepH profile is modified in the pH-range of 3.5-5.5. Still more inparticular, the activity at pH 4.0, 4.5, 5.0, and/or 5.5 is at a levelof at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or at least 95%of the activity at the pH-optimum.

Specific Activity

In a particular embodiment, the phytase of the invention has an improvedspecific activity relative to a reference phytase. More in particular,the specific activity of a phytase of the invention is at least 105%,relative to the specific activity of a reference phytase determined bythe same procedure. In still further particular embodiments, therelative specific activity is at least 110, 115, 120, 125, 130, 140,145, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 350 or even400%, still relative to the specific activity of the reference phytaseas determined by the same procedure.

In the alternative, the term high specific activity refers to a specificactivity of at least 200 FYT/mg Enzyme Protein (EP). In particularembodiments, the specific activity is at least 300, 400, 500, 600, 700,800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900,2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900 or 3000FYT/mg EP.

Specific activity is measured on highly purified samples (an SDS polyacryl amide gel should show the presence of only one component). Theenzyme protein concentration may be determined by amino acid analysis,and the phytase activity in the units of FYT, determined as described inExample 1. Specific activity is a characteristic of the specific phytasevariant in question, and it is calculated as the phytase activitymeasured in FYT units per mg phytase variant enzyme protein. See theExamples for further details.

In particular embodiments, an modified specific activity is expected ofthe following variants of the phytase of SEQ ID NO:2, in which, in orderof preference, the loop between replacing the loop between residues 115and 127 (HQQNTQQADPL) which faces the active site with a loop selectedfrom, e.g., HQEKMGTMDPT, HQQDIKQVDSL, HQPEIGKMDPV, TQADTSSPDPL,HQQDIKQADPL, TQTDTSSPDPL, NQADLKKTDPL.

Performance in Animal Feed

In a particular embodiment the phytase of the invention has an improvedperformance in animal feed as compared to a reference phytase. Theperformance in animal feed may be determined by the in vitro modelindicated in the Examples. Accordingly, in a preferred embodiment thephytase of the invention has an improved performance in animal feed,wherein the performance is determined in an in vitro model, by preparingfeed samples composed of 30% soybean meal and 70% maize meal with addedCaCl₂ to a concentration of 5 g calcium per kg feed; pre-incubating themat 40° C. and pH 3.0 for 30 minutes followed by addition of pepsin (3000U/g feed) and phytase; incubating the samples at 40° C. and pH 3.0 for60 minutes followed by pH 4.0 for 30 minutes; stopping the reactions;extracting phytic acid and inositol-phosphates by addition of HCl to afinal concentration of 0.5 M and incubation at 40° C. for 2 hours,followed by one freeze-thaw cycle and 1 hour incubation at 40° C.;separating phytic acid and inositol-phosphates by high performance ionchromatography; determining the amount of residual phytate phosphorus(IP6-P); calculating the difference in residual IP6-P between thephytase-treated and a non-phytase-treated blank sample (this differenceis degraded IP6-P); and expressing the degraded IP6-P of the phytase ofthe invention relative to degraded IP6-P of the reference phytase.

The phytase of the invention and the reference phytase are of coursedosed in the same amount, preferably based on phytase activity units(FYT). A preferred dosage is 125 FYT/kg feed. Another preferred dosageis 250 FYT/kg feed. The phytases may be dosed in the form of purifiedphytases, or in the form of fermentation supernatants. Purified phytasespreferably have a purity of at least 95%, as determined by SDS-PAGE.

In preferred embodiments, the degraded IP6-P value of the purifiedphytase of the invention, relative to the degraded IP6-P value of thereference phytase, is at least 101%, or at least 102%, 103%, 104%, 105%,110%, 115%, or at least 120%. In still further preferred embodiments,the degraded IP6-P value of the purified phytase of the invention,relative to the degraded IP6-P value of the reference phytase, is atleast 125%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or at least 200%.Preferably, the degraded IP6-P value of the phytase of the invention,relative to the degraded IP6-P value of the SEQ ID NO:2 phytase, is atleast 105%, 110%, 113%, 115%, 120%, 125%, or at least 130%.

The relative performance of a phytase of the invention may also becalculated as the percentage of the phosphorous released by thereference phytase.

In a still further particular embodiment, the relative performance ofthe phytase of the invention may be calculated as the percentage of thephosphorous released by the phytase of the invention, relative to theamount of phosphorous released by the reference phytase.

In still further particular embodiments, the relative performance of thephytase of the invention is at least 105%, preferably at least 110, 120,130, 140, 150, 160, 170, 180, 190, or at least 200%.

Reduction in Glycation

Nonenzymatic glycation is a spontaneous posttranslational process wherereducing sugars bind covalently to free amino groups in proteinsprimarily at Lysine (K) residues. In order to reduce glycation resultingin a reduction of activity of the phytase, the activity is improved bysubstituting certain amino acid residues, such as Lys. It is thereforeproposed to make one or more of the following modifications in thephytase of SEQ ID NO:2: K12R,Q, K26R,Q, K45P, K76R,Q, K97R,Q, K131R,Q,K139R,Q, K148R,Q, K176R,Q, K187E, K207L, K234R,Q, K251R,Q, K268R,Q,K299R,Q, K347R,Q, S261A, T308A, T25A, T28A, T30L, T219L, T120L, N₂₀₂A,N₂O₆A, N270R,Q, N312A, N119D, Q256A, Q29A, Q121A, Q122A, Q117A, Q118A,Y48W, and Y179L. Specifically preferred modifications in respect of thisimprovement in efficiency are the modifications K26Q and K26R.

Reduced Protease-Sensibility

In a particular embodiment, the phytase of the invention has a reducedprotease-sensibility. More in particular, it has a reduced sensibilitytowards the proteases pepsin and trypsin, meaning a reduced tendency tobecome cleaved by these protease.

The positions to be modified in this respect are indicated in Table 2below

TABLE 2 positions for modifying protease sensibility Pepsin PepsinTrypsin F8 W42 R22 L46 Y48 K26 L73 L74 K45 L112 F174 K76 L126 W237 K131L157 L323 R160 L188 L379 K176 W321 K187 L368 K234 L370 L395

To reduce the sensibility towards pepsin the amino acid residue shouldbe modified to an amino-acid different from F, L, W or Y. In the case ofTrypsin it should be modified to an amino acid residue different from Ror K.

Glycosylation Pattern

Glycosylation is a phenomenon which is only observed when expressingproteins in eukaryotes such as fungi and transgenic plants, but not inprokaryotes such as bacteria. There are various types of glycosylation,but in the present context the most relevant is the N-glycosylation,i.e., the asparagine-linked glycosylation where sugars are attached to aprotein, starting from an N-acetyglucosamine molecule attached toasparagines. N-glycosylation has been found to occur only to asparaginesthat in the sequence are part of the following tripeptides: N-X-T orN-X-S, where X designates any amino acid.

It has been observed that thermostability may be improved for phytasesexpressed in fungi by altering potential glycosylation sites.

The present invention accordingly also relates to phytase variantshaving an modified glycosylation pattern, preferably modifiedN-glycosylation sites. The modified glycosylation is expected to conferan improved thermostability upon the phytase variant, when expressed ina fungus.

Examples of phytases are bacterial phytases, e.g., Gram-negativephytases, such as E. coli, Citrobacter and Hafnia phytases and variantsthereof, including the phytases of the present invention. Examples offungal expression hosts are Pichia, Saccharomyces, and Aspergillusspecies.

In particular embodiments, an modified glycosylation pattern is expectedof the following phytases of the invention:

Removal of a glycosylation site. Res. number Res. type Change to 285 AsnAsp

New glycosylation sites: NXX Res. number Res. type Change to 121 GlnSer, Thr 186 Gly Ser, Thr 249 Leu Ser, Thr 331 Pro Ser, Thr 346 Gly Ser,Thr 355 Val Ser, Thr 382 Pro Ser, Thr

Creation of a new glycosylation sites of the type XXT Res. number Res.type Change to 33 Asp Asn 48 Tyr Asn 93 Gln Asn 96 Arg Asn 118 Gln Asn320 Thr Asn

Creation of a new glycosylation sites of the type XXS Res. number Res.type Change to 138 Asp Asn 162 Gln Asn 175 Pro Asn 190 Asp Asn 246 TrpAsn 293 Asp Asn 383 Gly Asn 394 Pro Asn 401 Leu Asn 403 Ser Asn

Steam Stability

Thermostability is an important parameter, but associated with that alsosteam stability is important. In this respect reference is made toExample 8 below.

Low-Allergenic Variants

In a specific embodiment, the phytases of the present invention are(also) low-allergenic variants, designed to invoke a reducedimmunological response when exposed to animals, including man. The termimmunological response is to be understood as any reaction by the immunesystem of an animal exposed to the phytase variant. One type ofimmunological response is an allergic response leading to increasedlevels of IgE in the exposed animal. Low-allergenic variants may beprepared using techniques known in the art. For example the phytasevariant may be conjugated with polymer moieties shielding portions orepitopes of the phytase variant involved in an immunological response.Conjugation with polymers may involve in vitro chemical coupling ofpolymer to the phytase variant, e.g., as described in WO 96/17929, WO98/30682, WO 98/35026, and/or WO 99/00489. Conjugation may in additionor alternatively thereto involve in vivo coupling of polymers to thephytase variant. Such conjugation may be achieved by genetic engineeringof the nucleotide sequence encoding the phytase variant, insertingconsensus sequences encoding additional glycosylation sites in thephytase variant and expressing the phytase variant in a host capable ofglycosylating the phytase variant, see e.g., WO 00/26354. Another way ofproviding low-allergenic variants is genetic engineering of thenucleotide sequence encoding the phytase variant so as to cause thephytase variants to self-oligomerize, effecting that phytase variantmonomers may shield the epitopes of other phytase variant monomers andthereby lowering the antigenicity of the oligomers. Such products andtheir preparation is described e.g., in WO 96/16177. Epitopes involvedin an immunological response may be identified by various methods suchas the phage display method described in WO 00/26230 and WO 01/83559, orthe random approach described in EP 561907. Once an epitope has beenidentified, its amino acid sequence may be altered to produce modifiedimmunological properties of the phytase variant by known genemanipulation techniques such as site directed mutagenesis (see e.g., WO00/26230, WO 00/26354 and/or WO 00/22103) and/or conjugation of apolymer may be done in sufficient proximity to the epitope for thepolymer to shield the epitope.

Nucleic Acid Sequences and Constructs

The present invention also relates to nucleic acid sequences comprisinga nucleic acid sequence which encodes a phytase variant of theinvention.

The term “isolated nucleic acid sequence” refers to a nucleic acidsequence which is essentially free of other nucleic acid sequences,e.g., at least about 20% pure, preferably at least about 40% pure, morepreferably at least about 60% pure, even more preferably at least about80% pure, and most preferably at least about 90% pure as determined byagarose electrophoresis. For example, an isolated nucleic acid sequencecan be obtained by standard cloning procedures used in geneticengineering to relocate the nucleic acid sequence from its naturallocation to a different site where it will be reproduced. The cloningprocedures may involve excision and isolation of a desired nucleic acidfragment comprising the nucleic acid sequence encoding the polypeptide,insertion of the fragment into a vector molecule, and incorporation ofthe recombinant vector into a host cell where multiple copies or clonesof the nucleic acid sequence will be replicated. The nucleic acidsequence may be of genomic, cDNA, RNA, semisynthetic, synthetic origin,or any combinations thereof.

The nucleic acid sequences of the invention can be prepared byintroducing at least one mutation into a template phytase codingsequence or a subsequence thereof, wherein the mutant nucleic acidsequence encodes a variant phytase. The introduction of a mutation intothe nucleic acid sequence to exchange one nucleotide for anothernucleotide may be accomplished by any of the methods known in the art,e.g., by site-directed mutagenesis, by random mutagenesis, or by doped,spiked, or localized random mutagenesis.

Random mutagenesis is suitably performed either as localized orregion-specific random mutagenesis in at least three parts of the genetranslating to the amino acid sequence shown in question, or within thewhole gene. When the mutagenesis is performed by the use of anoligonucleotide, the oligonucleotide may be doped or spiked with thethree non-parent nucleotides during the synthesis of the oligonucleotideat the positions which are to be changed. The doping or spiking may beperformed so that codons for unwanted amino acids are avoided. The dopedor spiked oligonucleotide can be incorporated into the DNA encoding thephytase enzyme by any technique, using, e.g., PCR, LCR or any DNApolymerase and ligase as deemed appropriate.

Preferably, the doping is carried out using “constant random doping”, inwhich the percentage of wild-type and mutation in each position ispredefined. Furthermore, the doping may be directed toward a preferencefor the introduction of certain nucleotides, and thereby a preferencefor the introduction of one or more specific amino acid residues. Thedoping may be made, e.g., so as to allow for the introduction of 90%wild type and 10% mutations in each position. An additionalconsideration in the choice of a doping scheme is based on genetic aswell as protein-structural constraints.

The random mutagenesis may be advantageously localized to a part of theparent phytase in question. This may, e.g., be advantageous when certainregions of the enzyme have been identified to be of particularimportance for a given property of the enzyme.

Alternative methods for providing variants of the invention include geneshuffling e.g., as described in WO 95/22625 or in WO 96/00343, and theconsensus derivation process as described in EP 897985.

Nucleic Acid Constructs

A nucleic acid construct comprises a nucleic acid sequence of thepresent invention operably linked to one or more control sequences whichdirect the expression of the coding sequence in a suitable host cellunder conditions compatible with the control sequences. Expression willbe understood to include any step involved in the production of thepolypeptide including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

The term “nucleic acid construct” as used herein refers to a nucleicacid molecule, either single- or double-stranded, which is isolated froma naturally occurring gene or which is modified to contain segments ofnucleic acids in a manner that would not otherwise exist in nature. Theterm nucleic acid construct is synonymous with the term “expressioncassette” when the nucleic acid construct contains the control sequencesrequired for expression of a coding sequence of the present invention.

The term “control sequences” is defined herein to include allcomponents, which are necessary or advantageous for the expression of apolynucleotide encoding a polypeptide of the present invention. Eachcontrol sequence may be native or foreign to the nucleotide sequenceencoding the polypeptide. Such control sequences include, but are notlimited to, a leader, polyadenylation sequence, propeptide sequence,promoter, signal peptide sequence, and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe nucleotide sequence encoding a polypeptide.

The term “operably linked” denotes herein a configuration in which acontrol sequence is placed at an appropriate position relative to thecoding sequence of the polynucleotide sequence such that the controlsequence directs the expression of the coding sequence of a polypeptide.

When used herein the term “coding sequence” (CDS) means a nucleotidesequence, which directly specifies the amino acid sequence of itsprotein product. The boundaries of the coding sequence are generallydetermined by an open reading frame, which usually begins with the ATGstart codon or alternative start codons such as GTG and TTG. The codingsequence may a DNA, cDNA, or recombinant nucleotide sequence

Expression Vector

The term “expression” includes any step involved in the production ofthe polypeptide including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

The term “expression vector” is defined herein as a linear or circularDNA molecule that comprises a polynucleotide encoding a polypeptide ofthe invention, and which is operably linked to additional nucleotidesthat provide for its expression.

A nucleic acid sequence encoding a phytase variant of the invention canbe expressed using an expression vector which typically includes controlsequences encoding a promoter, operator, ribosome binding site,translation initiation signal, and, optionally, a repressor gene orvarious activator genes.

The recombinant expression vector carrying the DNA sequence encoding aphytase variant of the invention may be any vector which mayconveniently be subjected to recombinant DNA procedures, and the choiceof vector will often depend on the host cell into which it is to beintroduced. The vector may be one which, when introduced into a hostcell, is integrated into the host cell genome and replicated togetherwith the chromosome(s) into which it has been integrated.

The phytase variant may also be co-expressed together with at least oneother enzyme of animal feed interest, such as a phytase, phosphatase,xylanase, galactanase, alpha-galactosidase, protease, phospholipase,amylase, and/or beta-glucanase. The enzymes may be co-expressed fromdifferent vectors, from one vector, or using a mixture of bothtechniques. When using different vectors, the vectors may have differentselectable markers, and different origins of replication. When usingonly one vector, the genes can be expressed from one or more promoters.If cloned under the regulation of one promoter (di- or multi-cistronic),the order in which the genes are cloned may affect the expression levelsof the proteins. The phytase variant may also be expressed as a fusionprotein, i.e., that the gene encoding the phytase variant has been fusedin frame to the gene encoding another protein. This protein may beanother enzyme or a functional domain from another enzyme.

Host Cells

The term “host cell”, as used herein, includes any cell type which issusceptible to transformation, transfection, transduction, and the likewith a nucleic acid construct comprising a polynucleotide of the presentinvention.

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention, which are advantageously usedin the recombinant production of the polypeptides. A vector comprising apolynucleotide of the present invention is introduced into a host cellso that the vector is maintained as a chromosomal integrant or as aself-replicating extra-chromosomal vector as described earlier. The term“host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the polypeptide and its source.

The host cell may be a unicellular microorganism, e.g., a prokaryote, ora non-unicellular microorganism, e.g., a eukaryote.

Useful unicellular microorganisms are bacterial cells such as grampositive bacteria including, but not limited to, a Bacillus cell, e.g.,Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacilluslautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium,Bacillus stearothermophilus, Bacillus subtilis, and Bacillusthuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans andStreptomyces murinus, or gram negative bacteria such as E. coli andPseudomonas sp. In a preferred aspect, the bacterial host cell is aBacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus, orBacillus subtilis cell. In another preferred aspect, the Bacillus cellis an alkalophilic Bacillus.

The introduction of a vector into a bacterial host cell may, forinstance, be effected by protoplast transformation (see, e.g., Chang andCohen, 1979, Molecular General Genetics 168: 111-115), using competentcells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower,1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5771-5278).

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

In a preferred aspect, the host cell is a fungal cell. “Fungi” as usedherein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota,and Zygomycota (as defined by Hawksworth et al., In, Ainsworth andBisby's Dictionary of The Fungi, 8th edition, 1995, CAB International,University Press, Cambridge, UK) as well as the Oomycota (as cited inHawksworth et al., 1995, supra, page 171) and all mitosporic fungi(Hawksworth et al., 1995, supra).

In a more preferred aspect, the fungal host cell is a yeast cell.“Yeast” as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980).

In an even more preferred aspect, the yeast host cell is a Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia cell.

In a most preferred aspect, the yeast host cell is a Pichia pastoris,Pichia methanolica, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomycesoviformis cell. In another most preferred aspect, the yeast host cell isa Kluyveromyces lactis cell. In another most preferred aspect, the yeasthost cell is a Yarrowia lipolytica cell.

In another more preferred aspect, the fungal host cell is a filamentousfungal cell. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra). The filamentous fungi are generally characterized by amycelial wall composed of chitin, cellulose, glucan, chitosan, mannan,and other complex polysaccharides. Vegetative growth is by hyphalelongation and carbon catabolism is obligately aerobic. In contrast,vegetative growth by yeasts such as Saccharomyces cerevisiae is bybudding of a unicellular thallus and carbon catabolism may befermentative.

In an even more preferred aspect, the filamentous fungal host cell is anAcremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,Coprinus, Coriolus, Cryptococcus, Filobasidium, Fusarium, Humicola,Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora,Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

In a most preferred aspect, the filamentous fungal host cell is anAspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus,Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger orAspergillus oryzae cell. In another most preferred aspect, thefilamentous fungal host cell is a Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusariumvenenatum cell. In another most preferred aspect, the filamentous fungalhost cell is a Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsisaneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens,Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa,Ceriporiopsis subvermispora, Coprinus cinereus, Coriolus hirsutus,Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthorathermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaetechrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris,Trametes villosa, Trametes versicolor, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride strain cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238 023 and Yelton et al., 1984, Proceedings of the NationalAcademy of Sciences USA 81: 1470-1474. Suitable methods for transformingFusarium species are described by Malardier et al., 1989, Gene 78:147-156, and WO 96/00787. Yeast may be transformed using the proceduresdescribed by Becker and Guarente, In Abelson, J. N. and Simon, M. I.,editors, Guide to Yeast Genetics and Molecular Biology, Methods inEnzymology 194: 182-187, Academic Press, Inc., New York; Ito et al.,1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978,Proceedings of the National Academy of Sciences USA 75: 1920.

Methods of Production

The present invention also relates to methods for producing a phytase ofthe present invention comprising (a) cultivating a host cell underconditions conducive for production of the phytase; and (b) recoveringthe phytase.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods well known in the art. For example, the cellmay be cultivated by shake flask cultivation, and small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The resulting polypeptide may be recovered using methods known in theart. For example, the polypeptide may be recovered from the nutrientmedium by conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989).

Transgenic Plants

The present invention also relates to a transgenic plant, plant part, orplant cell which has been transformed with a nucleotide sequenceencoding a polypeptide having phytase activity of the present inventionso as to express and produce the polypeptide in recoverable quantities.The polypeptide may be recovered from the plant or plant part.Alternatively, the plant or plant part containing the recombinantpolypeptide may be used as such for improving the quality of a food orfeed, e.g., improving nutritional value, palatability, and rheologicalproperties, or to destroy an antinutritive factor.

In a particular embodiment, the polypeptide is targeted to the endospermstorage vacuoles in seeds. This can be obtained by synthesizing it as aprecursor with a suitable signal peptide, see Horvath et al., 2000, PNAS97(4): 1914-1919.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot) or engineered variants thereof. Examples of monocot plantsare grasses, such as meadow grass (blue grass, Poa), forage grass suchas Festuca, Lolium, temperate grass, such as Agrostis, and cereals,e.g., wheat, oats, rye, barley, rice, sorghum, triticale (stabilizedhybrid of wheat (Triticum) and rye (Secale), and maize (corn). Examplesof dicot plants are tobacco, legumes, such as sunflower (Helianthus),cotton (Gossypium), lupins, potato, sugar beet, pea, bean and soybean,and cruciferous plants (family Brassicaceae), such as cauliflower, rapeseed, and the closely related model organism Arabidopsis thaliana.Low-phytate plants as described, e.g., in U.S. Pat. Nos. 5,689,054 and6,111,168 are examples of engineered plants.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers, as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyma, vascular tissues, meristems.Also specific plant cell compartments, such as chloroplast, apoplast,mitochondria, vacuole, peroxisomes, and cytoplasm are considered to be aplant part. Furthermore, any plant cell, whatever the tissue origin, isconsidered to be a plant part. Likewise, plant parts such as specifictissues and cells isolated to facilitate the utilisation of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seed coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts and plant cells.

The transgenic plant or plant cell expressing a polypeptide of thepresent invention may be constructed in accordance with methods known inthe art. Briefly, the plant or plant cell is constructed byincorporating one or more expression constructs encoding a polypeptideof the present invention into the plant host genome and propagating theresulting modified plant or plant cell into a transgenic plant or plantcell.

Conveniently, the expression construct is a nucleic acid construct whichcomprises a nucleic acid sequence encoding a polypeptide of the presentinvention operably linked with appropriate regulatory sequences requiredfor expression of the nucleic acid sequence in the plant or plant partof choice. Furthermore, the expression construct may comprise aselectable marker useful for identifying host cells into which theexpression construct has been integrated and DNA sequences necessary forintroduction of the construct into the plant in question (the latterdepends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences are determined, forexample, on the basis of when, where, and how the polypeptide is desiredto be expressed. For instance, the expression of the gene encoding apolypeptide of the present invention may be constitutive or inducible,or may be developmental, stage or tissue specific, and the gene productmay be targeted to a specific cell compartment, tissue or plant partsuch as seeds or leaves. Regulatory sequences are, for example,described by Tague et al., 1988, Plant Physiology 86: 506.

For constitutive expression, the following promoters may be used: The35S-CaMV promoter (Franck et al., 1980, Cell 21: 285-294), the maizeubiquitin 1 (Christensen, Sharrock and Quail, 1992, Maize polyubiquitingenes: structure, thermal perturbation of expression and transcriptsplicing, and promoter activity following transfer to protoplasts byelectroporation), or the rice actin 1 promoter (Plant Mo. Biol. 18:675-689; Zhang, McElroy. and Wu, 1991, “Analysis of rice Act1 5′ regionactivity in transgenic rice plants”, Plant Cell 3: 1155-1165).Organ-specific promoters may be, for example, a promoter from storagesink tissues such as seeds, potato tubers, and fruits (Edwards &Coruzzi, 1990, Ann. Rev. Genet. 24: 275-303), or from metabolic sinktissues such as meristems (Ito et al., 1994, Plant Mol. Biol. 24:863-878), a seed specific promoter such as the glutelin, prolamin,globulin, or albumin promoter from rice (Wu et al., 1998, Plant and CellPhysiology 39: 885-889), a Vicia faba promoter from the legumin B4 andthe unknown seed protein gene from Vicia faba (Conrad et al., 1998,Journal of Plant Physiology 152: 708-711), a promoter from a seed oilbody protein (Chen et al., 1998, Plant and Cell Physiology 39: 935-941),the storage protein napA promoter from Brassica napus, or any other seedspecific promoter known in the art, e.g., as described in WO 91/14772.Furthermore, the promoter may be a leaf specific promoter such as therbcs promoter from rice or tomato (Kyozuka et al., 1993, PlantPhysiology 102: 991-1000, the chlorella virus adenine methyltransferasegene promoter (Mitra and Higgins, 1994, Plant Molecular Biology 26:85-93), or the aldP gene promoter from rice (Kagaya et al., 1995,Molecular and General Genetics 248: 668-674), or a wound induciblepromoter such as the potato pin2 promoter (Xu et al., 1993, PlantMolecular Biology 22: 573-588). Likewise, the promoter may be inducibleby abiotic treatments such as temperature, drought or modifications insalinity or inducible by exogenously applied substances that activatethe promoter, e.g., ethanol, oestrogens, plant hormones like ethylene,abscisic acid, gibberellic acid, and/or heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of the polypeptide in the plant. For instance, the promoterenhancer element may be an intron which is placed between the promoterand the nucleotide sequence encoding a polypeptide of the presentinvention. For instance, Xu et al., 1993, supra disclose the use of thefirst intron of the rice actin 1 gene to enhance expression.

Still further, the codon usage may be optimized for the plant species inquestion to improve expression (see Horvath et al referred to above).

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38), andit can also be used for transforming monocots, although othertransformation methods are more often used for these plants. Presently,the method of choice for generating transgenic monocots, supplementingthe Agrobacterium approach, is particle bombardment (microscopic gold ortungsten particles coated with the transforming DNA) of embryonic callior developing embryos (Christou, 1992, Plant Journal 2: 275-281;Shimamoto, 1994, Current Opinion Biotechnology 5: 158-162; Vasil et al.,1992, Bio/Technology 10: 667-674). An alternative method fortransformation of monocots is based on protoplast transformation asdescribed by Omirulleh et al., 1993, Plant Molecular Biology 21:415-428.

Following transformation, the transformants having incorporated thereinthe expression construct are selected and regenerated into whole plantsaccording to methods well-known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using,e.g., co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating atransgenic plant or a plant cell comprising a nucleic acid sequenceencoding a polypeptide having phytase activity of the present inventionunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

Transgenic Animals

The present invention also relates to a transgenic, non-human animal andproducts or elements thereof, examples of which are body fluids such asmilk and blood, organs, flesh, and animal cells. Techniques forexpressing proteins, e.g., in mammalian cells, are known in the art, seee.g., the handbook Protein Expression: A Practical Approach, Higgins andHames (eds), Oxford University Press (1999), and the three otherhandbooks in this series relating to Gene Transcription, RNA processing,and Post-translational Processing. Generally speaking, to prepare atransgenic animal, selected cells of a selected animal are transformedwith a nucleic acid sequence encoding a polypeptide having phytaseactivity of the present invention so as to express and produce thepolypeptide. The polypeptide may be recovered from the animal, e.g.,from the milk of female animals, or the polypeptide may be expressed tothe benefit of the animal itself, e.g., to assist the animal'sdigestion. Examples of animals are mentioned below in the section headedAnimal Feed.

To produce a transgenic animal with a view to recovering the polypeptidefrom the milk of the animal, a gene encoding the polypeptide may beinserted into the fertilized eggs of an animal in question, e.g., by useof a transgene expression vector which comprises a suitable milk proteinpromoter, and the gene encoding the polypeptide. The transgeneexpression vector is microinjected into fertilized eggs, and preferablypermanently integrated into the chromosome. Once the egg begins to growand divide, the potential embryo is implanted into a surrogate mother,and animals carrying the transgene are identified. The resulting animalcan then be multiplied by conventional breeding. The polypeptide may bepurified from the animal's milk, see e.g., Meade, H. M. et al (1999):Expression of recombinant proteins in the milk of transgenic animals,Gene expression systems: Using nature for the art of expression. J. M.Fernandez and J. P. Hoeffler (eds.), Academic Press.

In the alternative, in order to produce a transgenic non-human animalthat carries in the genome of its somatic and/or germ cells a nucleicacid sequence including a heterologous transgene construct including atransgene encoding the polypeptide, the transgene may be operably linkedto a first regulatory sequence for salivary gland specific expression ofthe polypeptide, as disclosed in WO 00/064247.

Compositions and Uses

In still further aspects, the present invention relates to compositionscomprising a polypeptide of the present invention, as well as methods ofusing these.

The polypeptide compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. For instance, the polypeptide composition may be in theform of granulates or microgranulates. The polypeptide to be included inthe composition may be stabilized in accordance with methods known inthe art.

The phytase of the invention can be used for degradation, in anyindustrial context, of, for example, phytate, phytic acid, and/or themono-, di-, tri-, tetra- and/or penta-phosphates of myo-inositol. It iswell known that the phosphate moieties of these compounds chelatesdivalent and trivalent cations such as metal ions, i.a. thenutritionally essential ions of calcium, iron, zinc and magnesium aswell as the trace minerals manganese, copper and molybdenum. Besides,the phytic acid also to a certain extent binds proteins by electrostaticinteraction.

Accordingly, preferred uses of the polypeptides of the invention are inanimal feed preparations (including human food) or in additives for suchpreparations.

In a particular embodiment, the polypeptide of the invention can be usedfor improving the nutritional value of an animal feed. Non-limitingexamples of improving the nutritional value of animal feed (includinghuman food), are: Improving feed digestibility; promoting growth of theanimal; improving feed utilization; improving bio-availability ofproteins; increasing the level of digestible phosphate; improving therelease and/or degradation of phytate; improving bio-availability oftrace minerals; improving bio-availability of macro minerals;eliminating or reducing the need for adding supplemental phosphate,trace minerals, and/or macro minerals; and/or improving egg shellquality. The nutritional value of the feed is therefore increased, andthe growth rate and/or weight gain and/or feed conversion (i.e., theweight of ingested feed relative to weight gain) of the animal may beimproved.

Furthermore, the polypeptide of the invention can be used for reducingphytate level of manure.

The phytase variants of the invention can also be used in in a methodfor producing a fermentation product, comprising (a) fermenting using afermenting microorganism a carbohydrate containing material in thepresence of a phytase of the invention and (b) producing thefermentation product or fermentation coproduct from the fermentedcarbohydrate containing material.

When used for this purpose the fermentation product is preferablyethanol, beer, wine, or distillers dried grains (DDG).

Animals, Animal Feed, and Animal Feed Additives

The term animal includes all animals, including human beings. Examplesof animals are non-ruminants, and ruminants. Ruminant animals include,for example, animals such as sheep, goat, and cattle, e.g., cow such asbeef cattle and dairy cows. In a particular embodiment, the animal is anon-ruminant animal. Non-ruminant animals include mono-gastric animals,e.g., pig or swine (including, but not limited to, piglets, growingpigs, and sows); poultry such as turkeys, ducks and chickens (includingbut not limited to broiler chicks, layers); fish (including but notlimited to salmon, trout, tilapia, catfish and carp); and crustaceans(including but not limited to shrimp and prawn).

The term feed or feed composition means any compound, preparation,mixture, or composition suitable for, or intended for intake by ananimal.

In the use according to the invention the polypeptide can be fed to theanimal before, after, or simultaneously with the diet. The latter ispreferred.

In a particular embodiment, the polypeptide, in the form in which it isadded to the feed, or when being included in a feed additive, issubstantially pure. In a particular embodiment it is well-defined. Theterm “well-defined” means that the phytase preparation is at least 50%pure as determined by Size-exclusion chromatography (see Example 12 ofWO 01/58275). In other particular embodiments the phytase preparation isat least 60, 70, 80, 85, 88, 90, 92, 94, or at least 95% pure asdetermined by this method.

A substantially pure, and/or well-defined polypeptide preparation isadvantageous. For instance, it is much easier to dose correctly to thefeed a polypeptide that is essentially free from interfering orcontaminating other polypeptides. The term dose correctly refers inparticular to the objective of obtaining consistent and constantresults, and the capability of optimising dosage based upon the desiredeffect.

For the use in animal feed, however, the phytase polypeptide of theinvention need not be that pure; it may e.g., include otherpolypeptides, in which case it could be termed a phytase preparation.

The phytase preparation can be (a) added directly to the feed (or useddirectly in a treatment process of proteins), or (b) it can be used inthe production of one or more intermediate compositions such as feedadditives or premixes that is subsequently added to the feed (or used ina treatment process). The degree of purity described above refers to thepurity of the original polypeptide preparation, whether used accordingto (a) or (b) above.

Polypeptide preparations with purities of this order of magnitude are inparticular obtainable using recombinant methods of production, whereasthey are not so easily obtained and also subject to a much higherbatch-to-batch variation when the polypeptide is produced by traditionalfermentation methods.

Such polypeptide preparation may of course be mixed with otherpolypeptides.

The polypeptide can be added to the feed in any form, be it as arelatively pure polypeptide, or in admixture with other componentsintended for addition to animal feed, i.e., in the form of animal feedadditives, such as the so-called pre-mixes for animal feed.

In a further aspect the present invention relates to compositions foruse in animal feed, such as animal feed, and animal feed additives,e.g., premixes.

Apart from the polypeptide of the invention, the animal feed additivesof the invention contain at least one fat-soluble vitamin, and/or atleast one water soluble vitamin, and/or at least one trace mineral. Thefeed additive may also contain at least one macro mineral.

Further, optional, feed-additive ingredients are colouring agents, e.g.,carotenoids such as beta-carotene, astaxanthin, and lutein; aromacompounds; stabilisers; antimicrobial peptides; polyunsaturated fattyacids; reactive oxygen generating species; and/or at least one otherpolypeptide selected from amongst phytase (EC 3.1.3.8 or 3.1.3.26);phosphatase (EC 3.1.3.1; EC 3.1.3.2; EC 3.1.3.39); xylanase (EC3.2.1.8); galactanase (EC 3.2.1.89); alpha-galactosidase (EC 3.2.1.22);protease (EC 3.4.-.-), phospholipase A1 (EC 3.1.1.32); phospholipase A2(EC 3.1.1.4); lysophospholipase (EC 3.1.1.5); phospholipase C (3.1.4.3);phospholipase D (EC 3.1.4.4); amylase such as, for example,alpha-amylase (EC 3.2.1.1); and/or beta-glucanase (EC 3.2.1.4 or EC3.2.1.6).

In a particular embodiment these other polypeptides are well-defined (asdefined above for phytase preparations).

The phytase of the invention may also be combined with other phytases,for example ascomycete phytases such as Aspergillus phytases, forexample derived from Aspergillus ficuum, Aspergillus niger, orAspergillus awamori; or basidiomycete phytases, for example derived fromPeniophora lycii, Agrocybe pediades, Trametes pubescens, or Paxillusinvolutus; or derivatives, fragments or variants thereof which havephytase activity.

Thus, in preferred embodiments of the use in animal feed of theinvention, and in preferred embodiments of the animal feed additive andthe animal feed of the invention, the phytase of the invention iscombined with such phytases.

Examples of antimicrobial peptides (AMP's) are CAP18, Leucocin A,Tritrpticin, Protegrin-1, Thanatin, Defensin, Lactoferrin,Lactoferricin, and Ovispirin such as Novispirin (Robert Lehrer, 2000),Plectasins, and Statins, including the compounds and polypeptidesdisclosed in WO 03/044049 and WO 03/048148, as well as variants orfragments of the above that retain antimicrobial activity.

Examples of antifungal polypeptides (AFP's) are the Aspergillusgiganteus and Aspergillus niger peptides, as well as variants andfragments thereof which retain antifungal activity, as disclosed in WO94/01459 and WO 02/090384.

Examples of polyunsaturated fatty acids are C18, C20 and C22polyunsaturated fatty acids, such as arachidonic acid, docosohexaenoicacid, eicosapentaenoic acid and gamma-linoleic acid.

Examples of reactive oxygen generating species are chemicals such asperborate, persulphate, or percarbonate; and polypeptides such as anoxidase, an oxygenase or a syntethase.

Usally fat- and water-soluble vitamins, as well as trace minerals formpart of a so-called premix intended for addition to the feed, whereasmacro minerals are usually separately added to the feed. Either of thesecomposition types, when enriched with a polypeptide of the invention, isan animal feed additive of the invention.

In a particular embodiment, the animal feed additive of the invention isintended for being included (or prescribed as having to be included) inanimal diets or feed at levels of 0.01 to 10.0%; more particularly 0.05to 5.0%; or 0.2 to 1.0% (% meaning g additive per 100 g feed). This isso in particular for premixes.

The following are non-exclusive lists of examples of these components:

Examples of fat-soluble vitamins are vitamin A, vitamin D3, vitamin E,and vitamin K, e.g., vitamin K3.

Examples of water-soluble vitamins are vitamin B12, biotin and choline,vitamin B1, vitamin B2, vitamin B6, niacin, folic acid andpanthothenate, e.g., Ca-D-panthothenate.

Examples of trace minerals are manganese, zinc, iron, copper, iodine,selenium, and cobalt.

Examples of macro minerals are calcium, phosphorus and sodium.

The nutritional requirements of these components (exemplified withpoultry and piglets/pigs) are listed in Table A of WO 01/58275.Nutritional requirement means that these components should be providedin the diet in the concentrations indicated.

In the alternative, the animal feed additive of the invention comprisesat least one of the individual components specified in Table A of WO01/58275. At least one means either of, one or more of, one, or two, orthree, or four and so forth up to all thirteen, or up to all fifteenindividual components. More specifically, this at least one individualcomponent is included in the additive of the invention in such an amountas to provide an in-feed-concentration within the range indicated incolumn four, or column five, or column six of Table A.

The present invention also relates to animal feed compositions. Animalfeed compositions or diets have a relatively high content of protein.Poultry and pig diets can be characterised as indicated in Table B of WO01/58275, columns 2-3. Fish diets can be characterised as indicated incolumn 4 of this Table B. Furthermore such fish diets usually have acrude fat content of 200-310 g/kg.

WO 01/58275 corresponds to U.S. Ser. No. 09/779,334 which is herebyincorporated by reference.

An animal feed composition according to the invention has a crudeprotein content of 50-800 g/kg, and furthermore comprises at least onepolypeptide as claimed herein.

Furthermore, or in the alternative (to the crude protein contentindicated above), the animal feed composition of the invention has acontent of metabolisable energy of 10-30 MJ/kg; and/or a content ofcalcium of 0.1-200 g/kg; and/or a content of available phosphorus of0.1-200 g/kg; and/or a content of methionine of 0.1-100 g/kg; and/or acontent of methionine plus cysteine of 0.1-150 g/kg; and/or a content oflysine of 0.5-50 g/kg.

In particular embodiments, the content of metabolisable energy, crudeprotein, calcium, phosphorus, methionine, methionine plus cysteine,and/or lysine is within any one of ranges 2, 3, 4 or 5 in Table B of WO01/58275 (R. 2-5).

Crude protein is calculated as nitrogen (N) multiplied by a factor 6.25,i.e., Crude protein (g/kg)=N (g/kg)×6.25. The nitrogen content isdetermined by the Kjeldahl method (A.O.A.C., 1984, Official Methods ofAnalysis 14th ed., Association of Official Analytical Chemists,Washington D.C.).

Metabolisable energy can be calculated on the basis of the NRCpublication Nutrient requirements in swine, ninth revised edition 1988,subcommittee on swine nutrition, committee on animal nutrition, board ofagriculture, national research council. National Academy Press,Washington, D.C., pp. 2-6, and the European Table of Energy Values forPoultry Feed-stuffs, Spelderholt centre for poultry research andextension, 7361 DA Beekbergen, The Netherlands. Grafisch bedrijf Ponsen& looijen by, Wageningen. ISBN 90-71463-12-5.

The dietary content of calcium, available phosphorus and amino acids incomplete animal diets is calculated on the basis of feed tables such asVeevoedertabel 1997, gegevens over chemische samenstelling,verteerbaarheid en voederwaarde van voedermiddelen, CentralVeevoederbureau, Runderweg 6, 8219 pk Lelystad. ISBN 90-72839-13-7.

In a particular embodiment, the animal feed composition of the inventioncontains at least one protein. The protein may be an animal protein,such as meat and bone meal, and/or fish meal; or it may be a vegetableprotein. The term vegetable proteins as used herein refers to anycompound, composition, preparation or mixture that includes at least oneprotein derived from or originating from a vegetable, including modifiedproteins and protein-derivatives. In particular embodiments, the proteincontent of the vegetable proteins is at least 10, 20, 30, 40, 50, or 60%(w/w).

Vegetable proteins may be derived from vegetable protein sources, suchas legumes and cereals, for example materials from plants of thefamilies Fabaceae (Leguminosae), Cruciferaceae, Chenopodiaceae, andPoaceae, such as soy bean meal, lupin meal and rapeseed meal.

In a particular embodiment, the vegetable protein source is materialfrom one or more plants of the family Fabaceae, e.g., soybean, lupine,pea, or bean.

In another particular embodiment, the vegetable protein source ismaterial from one or more plants of the family Chenopodiaceae, e.g.,beet, sugar beet, spinach or quinoa.

Other examples of vegetable protein sources are rapeseed, sunflowerseed, cotton seed, and cabbage.

Soybean is a preferred vegetable protein source.

Other examples of vegetable protein sources are cereals such as barley,wheat, rye, oat, maize (corn), rice, triticale, and sorghum.

In still further particular embodiments, the animal feed composition ofthe invention contains 0-80% maize; and/or 0-80% sorghum; and/or 0-70%wheat; and/or 0-70% Barley; and/or 0-30% oats; and/or 0-40% soybeanmeal; and/or 0-25% fish meal; and/or 0-25% meat and bone meal; and/or0-20% whey.

Animal diets can, e.g., be manufactured as mash feed (non pelleted) orpelleted feed or extruded feed. Typically, the milled feed-stuffs aremixed and sufficient amounts of essential vitamins and minerals areadded according to the specifications for the species in question.Polypeptides can be added as solid or liquid polypeptide formulations.For example, a solid polypeptide formulation is typically added beforeor during the mixing step; and a liquid polypeptide preparation istypically added after the pelleting step. The polypeptide may also beincorporated in a feed additive or premix.

The final polypeptide concentration in the diet is within the range of0.01-200 mg polypeptide protein per kg diet, for example in the range of5-30 mg polypeptide protein per kg animal diet.

The phytase of the invention should of course be applied in an effectiveamount, i.e., in an amount adequate for improving solubilisation and/orimproving nutritional value of feed. It is at present contemplated thatthe polypeptide is administered in one or more of the following amounts(dosage ranges): 0.01-200; 0.01-100; 0.5-100; 1-50; 5-100; 10-100;0.05-50; or 0.10-10—all these ranges being in mg phytase polypeptideprotein per kg feed (ppm).

For determining mg phytase polypeptide protein per kg feed, the phytaseis purified from the feed composition, and the specific activity of thepurified phytase is determined using a relevant assay. The phytaseactivity of the feed composition as such is also determined using thesame assay, and on the basis of these two determinations, the dosage inmg phytase protein per kg feed is calculated.

The same principles apply for determining mg phytase polypeptide proteinin feed additives. Of course, if a sample is available of the phytaseused for preparing the feed additive or the feed, the specific activityis determined from this sample (no need to purify the phytase from thefeed composition or the additive).

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

EXAMPLES

Chemicals used were commercial products of at least reagent grade.

Example 1 Preparation of Variants, and Determination of ActivityPreparation of Phytase Variants

Expression of Phytase Variants in Asperqillus oryzae

The constructs comprising the Hafnia phytase variant genes in theexamples were used to construct expression vectors for Aspergillus. TheAspergillus expression vectors consist of an expression cassette basedon the Aspergillus niger neutral amylase II promoter fused to theAspergillus nidulans triose phosphate isomerase non translated leadersequence (Pna2/tpi) and the Aspergillus niger amyloglycosidaseterminator (Tamg). Also present on the plasmid was the Aspergillusselective marker amdS from Aspergillus nidulans enabling growth onacetamide as sole nitrogen source. The expression plasmids for phytasevariants were transformed into Aspergillus as described in Lassen etal., 2001, Applied and Environmental Microbiology 67: 4701-4707. Foreach of the constructs 10-20 strains were isolated, purified andcultivated in shake flasks.

Purification of Hafnia alvei Phytase Variants

The fermentation supernatant with the phytase variant was filteredthrough a Fast PES Bottle top filter with a 0.22 μm cut-off. Theresulting solution was diluted with water to the double volume and pHwas adjusted to 4.5 with acetic acid. Occasionally, the solution becamea little cloudy and this removed by filtration through a Fast PES Bottletop filter with a 0.22 μm cut-off.

After pretreatment the phytase variant was purified by chromatography onS Sepharose, approximately 30 ml in a XK26 column, using as buffer A 50mM sodium acetate pH 4.5, and as buffer B 50 mM sodium acetate+1 M NaClpH 4.5. The fractions from the column were analyzed for activity usingthe phosphatase assay (see below) and fractions with activity werepooled.

Finally, the solution containing the purified phytase variant wasconcentrated using an Amicon ultra-15 filtering device with a 30 kDacut-off membrane.

The molecular weight, as estimated from SDS-PAGE, was approximately 45kDa and the purity was >95%.

Determination of Phosphatase Activity

75 microliters phytase-containing enzyme solution is dispensed in amicrotiter plate well, e.g. NUNC 269620 and 75 microliter substrate isadded (for preparing the substrate, two 5 mg p-nitrophenyl phosphatetablets (Sigma, Cat. No. N-9389) are dissolved in 10 ml 0.1 M Na-acetatebuffer, pH 5.5). The plate is sealed and incubated 15 min., shaken with750 rpm at 37° C. After the incubation time 75 microliter stop reagentis added (the stop reagent is 0.1 M di-sodiumtetraborate in water) andthe absorbance at 405 nm is measured in a microtiter platespectrophotometer. One phosphatase unit is defined as the enzymeactivity that releases 1 micromol phosphate/min under the given reactionconditions (buffer blind subtracted). The absorbance of 1 micromolp-nitrophenol is determined to be 56 AU (AU=absorbancy units) underassay conditions.

Determination of Phytase Activity

75 microliters phytase-containing enzyme solution, appropriately dilutedin 0.25 M sodium acetate, 0.005% (w/v) Tween-20. pH 5.5, is dispensed ina microtiter plate well, e.g. NUNC 269620, and 75 microliter substrateis added (prepared by dissolving 100 mg sodium phytate from rice(Aldrich Cat. No. 274321) in 10 ml 0.25 M sodium acetate buffer, pH5.5). The plate is sealed and incubated 15 min. shaken with 750 rpm at37° C. After incubation, 75 microliters stop reagent is added (the stopreagent being prepared by mixing 10 ml molybdate solution (10% (w/v)ammonium hepta-molybdate in 0.25% (w/v) ammonia solution), 10 mlammonium vanadate (0.24% commercial product from Bie&Berntsen, Cat. No.LAB17650), and 20 ml 21.7% (w/v) nitric acid), and the absorbance at 405nm is measured in a microtiter plate spectrophotometer. The phytaseactivity is expressed in the unit of FYT, one FYT being the amount ofenzyme that liberates 1 micromole inorganic ortho-phosphate per minuteunder the conditions above. An absolute value for the measured phytaseactivity may be obtained by reference to a standard curve prepared fromappropriate dilutions of inorganic phosphate, or by reference to astandard curve made from dilutions of a phytase enzyme preparation withknown activity (such standard enzyme preparation with a known activityis available on request from Novozymes NS, Krogshoejvej 36, DK-2880Bagsvaerd).

Example 2 Specific Activity

The specific activity of a phytase variant is determined on highlypurified samples dialysed against 250 mM sodium acetate, pH 5.5. Thepurity is checked beforehand on an SDS poly acryl amide gel showing thepresence of only one component.

The protein concentration is determined by amino acid analysis asfollows: An aliquot of the sample is hydrolyzed in 6 N HCl, 0.1% phenolfor 16 h at 110° C. in an evacuated glass tube. The resulting aminoacids are quantified using an Applied Biosystems 420A amino acidanalysis system operated according to the manufacturer's instructions.From the amounts of the amino acids the total mass—and thus also theconcentration—of protein in the hydrolyzed aliquot can be calculated.

The phytase activity is determined in the units of FYT as described inExample 1 (“Determination of phytase activity”), and the specificactivity is calculated as the phytase activity measured in FYT units permg phytase variant enzyme protein.

Example 3 Temperature Stability Strains and Plasmids

E. coli DH12S (available from Gibco BRL) was used for yeast plasmidrescue.

pJHP000 is a S. cerevisiae and E. coli shuttle vector under the controlof TPI promoter, constructed from pJC039 described in WO 01/92502, inwhich the Hafnia alvei phytase gene has been inserted.

Saccharomyces cerevisiae YNG318: MATa Dpep4[cir+] ura3-52, leu2-D2, h is4-539 was used for the phytase variants expression. It is described inJ. Biol. Chem. 272(15): 9720-9727, 1997.

Media and Substrates

10× Basal solution: Yeast nitrogen base w/o amino acids (DIFCO) 66.8g/l, succinate 100 g/l, NaOH 60 g/l.

SC-glucose: 20% glucose (i.e., a final concentration of 2%=2 g/100 ml))100 ml/l, 5% threonine 4 ml/l, 1% tryptophan 10 ml/l, 20% casamino acids25 ml/l, 10× basal solution 100 ml/l. The solution is sterilized using afilter of a pore size of 0.20 micrometer. Agar and H2O (approx. 761 ml)is autoclaved together, and the separately sterilized SC-glucosesolution added to the agar solution.

YPD: Bacto peptone 20 g/l, yeast extract 10 g/l, 20% glucose 100 ml/l.

PEG/LiAc solution: 40% PEG4000 50 ml, 5 M Lithium Acetate 1 ml

DNA Manipulations

Unless otherwise stated, DNA manipulations and transformations wereperformed using standard methods of molecular biology as described inSambrook et al., 1989, Molecular cloning: A laboratory manual, ColdSpring Harbor lab. Cold Spring Harbor, N.Y.; Ausubel, F. M. et al.(eds.) “Current protocols in Molecular Biology”, John Wiley and Sons,1995; Harwood, C. R. and Cutting, S. M. (eds.).

Yeast Transformation

Yeast transformation was carried out by lithium acetate method. Mix 0.5microL of vector (digested by restriction endonucleases) and 1 microL ofPCR fragments. Thaw YNG318 competent cells on ice. Mix 100 microL of thecells, the DNA mixture and 10 microL of carrier DNA (Clontech) in 12 mlpolypropylene tubes (Falcon 2059). Add 0.6 ml PEG/LiAc solution and mixgently. Incubate for 30 min at 30° C., and 200 rpm. Incubate for 30 minat 42° C. (heat shock). Transfer to an Eppendorf tube and centrifuge for5 sec. Remove the supernatant and resolve in 3 ml of YPD. Incubate thecell suspension for 45 min at 200 rpm at 30° C. Pour the suspension toSC-glucose plates and incubate 30° C. for 3 days to make colonies. Yeasttotal DNA was extracted by the Robzyk and Kassir's method described inNucleic Acids Research 20(14): 3790 (1992).

DNA Sequencing

E. coli transformation for DNA sequencing was carried out byelectroporation (BIO-RAD Gene Pulser). DNA Plasmids were prepared byalkaline method (Molecular Cloning, Cold Spring Harbor) or with theQiagen® Plasmid Kit. DNA fragments were recovered from agarose gel bythe Qiagen gel extraction Kit. PCR was performed using a PTC-200 DNAEngine. The ABI PRISM™ 310 Genetic Analyzer was used for determinationof all DNA sequences.

Construction of Phytase Expression Vector The Hafnia phytase gene wasamplified with the primer pairs (HafPhyF and HafPHyR). The resulting PCRfragments were introduced into S. cerevisiae YNG318 together with thepJC039 vector digested with restriction enzymes to remove the maturepart of Humicola insolens cutinase gene.

HafPhyF (34mer) CTCCTGAACTTGTTGCCCGGTCGGATACAGCCCC HafPhyR (39mer)ATTACATGATGCGGCCCTCTAGATTAGGGGAGCTGACATG

Plasmid, which is termed as pJHP000 from the yeast transformants onSC-glucose plates was recovered and the internal sequence was determinedto confirm the phytase gene.

Construction of Yeast Library and Site-Directed Variants

Library in yeast and site-directed variants were constructed by SOE PCRmethod (Splicing by Overlap Extension, see “PCR: A practical approach”,p. 207-209, Oxford University press, eds. McPherson, Quirke, Taylor),followed by yeast in vivo recombination.

General Primers for Amplification and Sequencing

The below primers are used to make DNA fragments containing any mutatedfragments by the SOE method together with degenerated primers(AM34+Reverse primer and AM35+forward primer) or just to amplify a wholeasparaginase gene (AM34+AM35).

AM34 TAGGAGTTTAGTGAACTTGC AM35 TTCGAGCGTCCCAAAACC PCR reaction system:Conditions: 48.5 micro L H₂O 1 94° C.  2 min 2 beads puRe Taq Ready-To-2 94° C. 30 sec Go PCR Beads (Amersham bioscineces) 3 55° C. 30 sec0.5 micro L X 2100 pmole/micro L 4 72° C. 90 sec Primers 0.5 micro LTemplate DNA 2-4 25 cycles 5 72° C. 10 min

DNA fragments were recovered from agarose gel by the Qiagen gelextraction Kit. The resulting purified fragments were mixed with thevector digest. The mixed solution was introduced into Saccharomycescerevisiae to construct libraries or site-directed variants by in vivorecombination.

Library Screening (the Primary Membrane Assay)

Yeast libraries were cultivated on SC-glucose plate with a celluloseacetate membrane (upper) and Biodyne C (from Pall gelman) membrane(lower) at 30° C. at least for 3 days. The BiodyneC membranes weretransferred to pre-incubated plates containing 20 mM acetate buffer, pH4.0 and incubated for 1-2 hours at a certain temperature (50° C. in thecase of WT as a backbone).

Then, the membranes were removed and soaked in the fresh substratesolution (10 ml 20 mM acetate buffer, pH 4.0; 0.01 g, alpha-naphtylphosphate (sigma); 0.02 g, Fast Garnet GBC (sigma)). Yeast clonescorresponding to the positions of red color developed on the Biodyne Cmembranes were isolated from cellulose acetate membranes.

Library Screening (the Secondary Relative Activity Selection)

Yeast clones on cellulose acetate membranes were inoculated to a well ofa 96-well micro titre plate and cultivated at 28° C. for 3 days. Phytaseactivity was measured at both 37° C. and the higher temperature (60, 62,64, 66° C. etc.) to determine the relative activity at a certaintemperature. Then the clones with higher relative activity were selectedand the sequence was confirmed.

Standard, Level control and samples are pipetted 10 microliters into aMTP or 8 strip tube. Pre-heated (50° C.) substrate is added. 200microliters The 8-stripe tube or MTP is placed in an MTP 30 min.incubator at 37, 60 and 64° C. (or above). Take out 35 μl, add it into100 microliters of stop- 35 + 100 microliters complex reagent and mixed5-20 s. The sample waits before measurement. 5-30 min OD is measured at750 nm

Substrate, Sodium Phytate Solution 2.0 mM (Every Time)

Example of preparation of 100 ml:Sodium phytate 0.1847 g0.1 M Acetate buffer, pH4.0 up to 100 ml

Complexing Reagent

Example of preparation of 200 ml:

FeSO₄.7H₂O 14.64 g

Ammonium heptamolybdate solution up to 200 ml

Stop-Complex Reagent

Example of preparation of 600 ml stop-complex reagent

0.5 M H2SO4 200 ml Complexing Reagent 400 ml

Ammonium heptamolybdate solutionExample of preparation of 1000 ml:

(NH₄)₆Mo7O₂₄.4H₂O 10.0 g

Sulfuric acid 32 mlDemineralized water up to 1000 ml

The results are provided below. The column indicating the relativeactivity provides first the relative activity of the variant andthereafter the relative activity of the reference phytase used in thedetermination. The reference is either the wild type or variants nos.62, 69, 84, 104, 105, 113 and/or 138. The variants were typically usedas reference when the residual activity of the wild type was very low.

Results

Relative activity (control/reference Variant no. Modifications(substitutions, insertions or deletions) variant) 005 A132V/Q162R 11% at72° C. (WT2%) 007 Y179W 12% at 72° C. (WT2%) 008 A132V/Q181L 15% at 72°C. (WT2%) 011 A132V/E211R 10% at 72° C. (WT2%) 012 A132V/D83G 11% at 72°C. (WT3%) 013 A132V/A217G 50% at 72° C. (WT2%) 014 A132V/A217S 47% at72° C. (WT2%) 015 A221G 11% at 72° C. (WT2%) 016 R32I 46% at 72° C.(WT26%) 017 R32L 38% at 72° C. (WT26%) 020 D77G 46% at 72° C. (WT26%)021 D77W 40% at 72° C. (WT26%) 023 T95A 44% at 72° C. (WT26%) 024E100W/H363R 42% at 72° C. (WT26%) 025 D111S 69% at 72° C. (WT26%) 027D138V/Y48H 48% at 72° C. (WT39%) 028-1 K234C 50% at 72° C. (WT39%) 028-2K234V 50% at 72° C. (WT39%) 029 K251S 46% at 72° C. (WT39%) 030 H363V40% at 72° C. (WT39%) 031 H363R 53% at 72° C. (WT39%) 046-1 A132V/A217G42% at 70° C. (WT13%) 046-2 A132V/Q162R/Q181L/A217G 42% at 70° C.(WT13%) 047 D293R 30% at 70° C. (WT13%) 048 Q93E 22% at 70° C. (WT13%)050-1 P348R/H363R 30% at 70° C. (WT13%) 050-2 P348S 30% at 70° C.(WT13%) 051 Q69L 20% at 70° C. (WT13%) 052 Q245E 19% at 70° C. (WT13%)053 Q9S/D92Y 42% at 70° C. (WT13%) 054-2 D92Y/H115L 28% at 70° C.(WT13%) 054-re1,2 D92Y/H115M 32% at 70° C. (WT17%) 057 N78Q 56% at 70°C. (WT28%) 058 K76V 57% at 70° C. (WT28%) 061-1 G325K 47% at 70° C.(WT28%) 062 E100W/A217G/H363R(reference) 63% at 72° C. (WT17%) 063A217G/K251S 40% at 72° C. (WT17%) 064 E100W/A217G/K251S 38% at 72° C.(WT17%) 065 E100W/K251S 26% at 72° C. (WT17%) 066 A217G 29% at 72° C.(WT10%) 067 E100W/I555V/A217G 45% at 72° C. (WT9%) 068Q9S/E100W/R160G/A217G/H363R 45% at 72° C. (WT9%) 069D92Y/E100W/A217G/H363R(reference) 79% at 72° C. (WT9%) 070E100W/H115M/A217G/H363R 41% at 72° C. (WT9%) 071 E100W/A217G/P348R/H363R63% at 72° C. (WT9%) 072 Q9S/A89A/D92Y/H115M/A217G/H363R 67% at 72° C.(WT9%) 073 A132T 21% at 72° C. (WT16%) Reference = variant no. 62 075N78Q/E100W/A217G/H363R 47% at 72° C. (62 40%) 076K76V/N78Q/E100W/A217G/H363R 47% at 72° C. (62 40%) 077D83G/E100W/A217G/H363R 39% at 72° C. (62 40%) 078E100W/Y179W/A217G/H363R 48% at 72° C. (62 40%) 079E100W/A217G/K234V/K251E/I286T/H363R 60% at 72° C. (62 40%) 081E100W/A217G/K234V/P348R/H363R 50% at 72° C. (62 35%) 082Q9S/R18K/A89A/D92Y/H115M/A217G/K234V/ 61% at 72° C. (62 35%) H363R082v2-1 Q9S/D92Y/H115M/A217G/K234V/H363R 61% at 72° C. (62 35%) 083Q9S/N78Q/D92Y/L112S/H115M/K234V/P348R/ 39% at 72° C. (62 35%) H363R 084Q9S/N78Q/A89A/D92Y/H115M/A132V/Q162R/ 64% at 72° C. (62 35%)Q181L/A217G/K234V/P348R(reference) Reference = WT and/or variant no. 69085 Q9S/E54C/D92Y/A101C/H143C/Q193R/I201C/ 82% at 72° C. (69 74%A217G/H363R WT 14%) 086 E54C/N78S/D92Y/A101C/H143C/L199C/A217G/ 74% at72° C. (wt 49% 69 H363R 80%) 088 E54C/A101C/M168V/A217G/H363R 33% at 72°C. (62 36% 69 73%) 089-1 P82S/D92Y/E100W/H143C/I201C/A217G/H363R 65% at72° C. (62 36% 69 73%) 089-2 P82S/D92Y/E100W/H143C/I201C/A217G/H363R 65%at 72° C. (62 36% 69 73%) 090 Q9S/N78Q/D92Y/L112S/H115M/A217G/K234V/ 65%at 72° C. (62 36% 69 P348R/H363R 73%) 091 D92Y/A217G/K234V/H363R 81% at72° C. (62 36% 69 73%) 092-1 Y64S/D92Y/E100W/Y179W/A217G/H363R 80% at72° C. (69 74% WT 14%) 092-2 D92Y/A217G/H363R 80% at 72° C. (69 74% WT14%) 094 Q9S/N78Q/A89A/D92Y/H115M/A132V/H143C/ 54% at 74° C. (wt 9% 69Q162R/Q181L/I201C/A217G/K234V/P348R 45%) 095Q9S/N78Q/A89A/D92Y/H115M/A132V/K139C/ 75% at 74° C. (wt 9% 69Q162R/Q181L/I201C/A217G/K234V/P348R 45%) 097Q9S/N78Q/A89A/D92Y/H115M/A132V/Q162R/ 76% at 74° C. (wt 9% 69Y179W/Q181L/A217G/K234V/P348R 45%) 100D33C/D92Y/E100W/Y179C/A217G/H363R/ 49% at 72° C. (69 79%) 103-1Q9S/N78Q/A89A/D92Y/H115M/A132V/Q162R/ 103% at 72° C. (69 88%)Y179W/A217G/K234V/P348R/H363R 103-3Q9S/N78Q/A89A/D92Y/H115M/A132V/Q162R/ 99% at 72° C. (69 88%)Y179W/A217G/K234V/S261F/P348R/H363R Reference = variant 69 and/orvariant 84 101 D92Y/E100W/A217G/H363R/+ 116-123(HQQNTQQA 21% at 76° C.(69 39%, ->TQADTSSP) 84 55%) 102 Q9S/E54C/D92Y/A101C/H143C/Q193R/I201C/29% at 76° C. (69 39%, A217G/N298S/H363R +116-123(HQQNTQQA-> 84 55%)TQADTSSP) 104 Q9S/N78Q/A89A/D92Y/H115M/A132V/K139C/ 55% at 74° C. (6957% 84 G151D/Q162R/Y179W/Q181L/I201C/A217G/K234V/ 65%) P348R (reference)105 D92Y/E100W/K139C/I201C/A217G/N247D/H363R 77% at 74° C. (69 57% 84(reference) 65%) Reference = variant 104 and variant 69 106E54C/D92Y/A101C/M168V/A217G/H363R 22% at 74° C. (104 70% 69 54%) 107Q9S/N78Q/A132V/K139C/Q162R/Y179W/I201C/ 34% at 74° C. (104 70%A217G/K234L/P348R/H363R 69 54%) Reference = variant 105 108D92Y/E100W/H143C/A144R/I201C/A217G/N247D/ 37% at 80° C. (105 36%) H363R109 D92Y/E100W/H116S/K139C/I201C/A217G/N247D/ 43% at 78° C. (105 36%)H363R 110 D92Y/E100W/H128R/K139C/H143V/I201C/A217G/ 44% at 78° C. (10536%) N247D/H363R 111 D92Y/E100W/K139C/I201C/N206G/A217G/N247D/ 45% at78° C. (105 36%) H363R 112 D92Y/E100W/K139C/I201C/A217G/N247D/H363R 51%at 78° C. (105 36%) 113 D92Y/E100W/K139C/I201C/A217G/N247D/Q256D/ 98% at78° C. (105 36%) H363R 114 D92Y/E100W/K139C/I201C/A217G/N247D/H363R 49%at 78° C. (105 36%) 115 D92Y/E100W/K139C/I201C/A217G/N247D/N344K/ 32% at78° C. (105 36%) H363R 117 D92Y/E100W/K139C/A144S/K176E/I201C/A217G/ 38%at 80° C. (105 34%) K234V/N247D/H363R 118D92Y/E100W/K139C/I201C/A217G/K234V/N247D/ 32% at 80° C. (105 34%)H363R/E54C/H55E/A101C 123 D92Y/E100W/K139C/T152A/I201C/A217G/K234V/ 27%at 80° C. (105 15%) N247D/H363R 124Y48H/D92Y/E100W/K139C/T152I/I201C/A217G/ 17% at 80° C. (105 15%)K234V/N247D/H363R 125 D92Y/E100W/K139C/I201C/A217G/K234V/N247D/ 20% at80° C. (105 15%) S284C/H363R 126D92Y/E100W/K139C/I201C/A217G/K234V/N247D/ 20% at 80° C. (105 15%)T287W/H363R 127 D92Y/E100W/K139C/I201C/A217G/K234V/N247D/ 18% at 80° C.(105 15%) R289M/H363R 128 Y48H/D92Y/E100W/K139C/I201C/A217G/K234V/ 17%at 80° C. (105 15%) N247D/R289W/H363R 129N78Q/D92Y/E100W/K139C/I201C/A217G/K234V/ 103% at 80° C. (105N247D/Q256D/H363R 34%) 130 D92Y/E100W/K139C/I201C/A217G/K234V/N247D/ 99%at 80° C. (105 34%) Q256D/P348R/H363R 131D92Y/E100W/K139C/Q162R/Q181L/I201C/A217G/ 76% at 80° C. (105 34%)K234V/N247D/Q256D/H363R 132 D92Y/E100W/A113G/K139C/I201C/A217G/K234V/34% at 80° C. (105 34%) N247D/H363R 133D92Y/E100W/T120GorA*/K139C/I201C/A217G/K234V/ 41% at 80° C. (105 34%)N247D/H363R/L395LorV 135 D92Y/E100W/K139C/I201C/A217G/K234V/N247D/ 24%at 80° C. (105 34%) S284M/H363R 137D92Y/E100W/K139C/I201C/A217G/K234V/N247D/ 31% at 80° C. (105 34%)H363R/A366S 138 D92Y/E100W/K139C/I201C/A217G/K234V/N247W/ 89% at 80° C.(105 14%) Q256D/H363R 140 D92Y/E100W/H128R/K139C/I201C/A217G/K234V/ 49%at 80° C. (105 14%) N247E/Q256D/H363R 141D92Y/E100W/K139C/Q141S/I201C/A217G/K234V/ 24% at 80° C. (105 14%)N247D/H363R 142 D92Y/E100W/K139C/A144S/I201C/A217G/K234V/ 18% at 80° C.(105 14%) N247D/H363R 144 P75N/K76N/D77Q/N78T/D92Y/E100W/K139C/I201C/71% at 80° C. (105 30%) A217G/K234V/N247D/Q256D/H363R 145D92Y/E100W/K139C/D173N/P175S/I201C/A217G/ 27% at 80° C. (105 30%)K234V/N247D/Q256D/H363R 147 D92Y/E100W/K139C/T152A/I201C/A217G/K234V/90% at 80° C. (105 26%) N247D/Q256D/I294T/H363R Reference = variant 113143 D33N/D92Y/E100W/K139C/I201C/A217G/K234V/ 29% at 80° C. (113 62%)N247D/Q256D/H363R 148 Y48H/D92Y/E100W/K139C/T152I/I201C/A217G/ 74% at80° C. (113 80%) K234V/N247D/Q256D/H363R 150D92Y/E100W/K139C/T152A/I201C/A217G/K234V/ 67% at 82° C. (113 33%)N247D/Q256D/H363R 151 D92Y/T98S/E100W/K139C/T152A/I201C/A217G/ 29% at82° C. (113 33%) K234V/N247D/Q256D/H363R 152D92Y/T98S/E100W/K139C/T152A/L199S/I201C/ 38% at 80° C. (113 80%)A217G/K234V/N247W/Q256D/H363R 153Y48H/D92Y/T98S/E100W/K139C/T152A/I201C/ 38% at 80° C. (113 54%)A217G/K234V/N247W/Q256D/H363R 154E54C/D92Y/A101C/K139C/I201C/A217G/K234V/ 91% at 80° C. (113 73%)N247D/Q256D/H363R 155 Y48H/E54C/D92Y/A101C/K139C/I201C/A217G/ 88% at 80°C. (113 80%) K234V/N247D/R289W/H363R 156D92Y/T98S/E100W/K139C/T152A/I201C/A217G/ 41% at 82° C. (113 33%)K234V/N247W/Q256D/R289W/H363R 157Y48H/D92/E100W/K139C/T152I/I201C/A217G/ 79% at 80° C. (113 80%)K234V/N247W/Q256D/R289W/H363R 158Y48H/D92Y/E100W/K139C/I201C/A217G/K234V/ 95% at 80° C. (113 80%)N247W/Q256D/H363R 159 Y48H/D92Y/E100W/K139C/T152A/I201C/A217G/ 20% at80° C. (113 57%) K234V/N247D/R289W/H363R 160Y48H/D92Y/E100W/K139C/T152A/I201C/A217G/ 92% at 80° C. (113 80%)K234V/N247W/Q256D/H363R 161 Y48H/E54C/D92Y/A101C/K139C/T152A/I201C/ 55%at 80° C. (113 80%) A217G/K234V/N247D/R289W/H363R 162Y48H/E54C/D92Y/A101C/K139C/I201C/A217G/ 56% at 80° C. (113 80%)K234V/N247W/R289W/H363R 163 Y48H/E54C/D92Y/A101C/K139C/T152A/I201C/ 66%at 80° C. (113 80%) A217G/K234V/N247W/R289W/H363R Reference = variant138 164 Y48H/E54C/D92Y/E100W/A101C/K139C/T152A/ 94% at 80° C. (138 78%)I201C/A217G/K234V/N247W/Q256D/H363R 165Y48H/E54C/D92Y/A101C/K139C/T152A/I201C/ 65% at 80° C. (138 78%)V208T/A217G/K234V/N247D/R289W/H363R 166T35A/Y48H/E54C/P75N/K76N/D77Q/N78T/D92Y/ 15% at 80° C. (138 78%)A101C/K139C/T152A/I201C/A217G/K234V/N247D/ R289W/H363R 167Y48H/E54C/D92Y/A101C/K139C/T152A/I201C/ 61% at 80° C. (138 78%)K207Q/V208T/A217G/K234V/N247D/R289W/H363R Reference = wt 168E54C/D92Y/A101C/K139C/I201C/A217G/Q256D/ 69% at 80° C. (w 7%) H363R 169E54C/P75N/K76N/D77Q/N78T/D92Y/A101C/K139C/ 33% at 80° C. (w 7%)I201C/A217G/H363R 170 E54C/D92Y/A101C/K139C/I201C/V208T/A217G/ 32% at80° C. (w 7%) H363R 171 E54C/P75N/K76N/D77Q/N78T/D92Y/A101C/K139C/ 36%at 80° C. (w 7%) I201C/V208T/A217G/H363R 172Y48H/E54C/P75N/K76N/D77Q/N78T/D92Y/A101C/ 48% at 80° C. (w 7%)K139C/T152A/I201C/V208T/A217G/K234V/N247D/ R289W/H363R 173E54C/P75N/K76N/D77Q/N78T/D92Y/A101C/K139C/ 50% at 80° C. (w 7%)I201C/V208T/A217G/K234V/N239S/N247D/Q256D/ H363R

Example 4 Thermostability

An aliquot of the protein sample of Hafnia alvei phytase (purified asdescribed in Example 1) was either desalted and buffer-changed into 20mM Na-acetate, pH 4.0 using a prepacked PD-10 column or dialysed against2×500 ml 20 mM Na-acetate, pH 4.0 at 4° C. in a 2-3 h step followed byan overnight step. The sample was 0.45 micro-m filtered and diluted withbuffer to approx. 2 A280 units. The dialysis buffer was used asreference in Differential Scanning calorimetry (DSC). The samples weredegassed using vacuum suction and stirring for approx. 10 minutes.

A DSC scan was performed on a MicroCal VP-DSC at a constant scan rate of1.5° C./min from 20-90° C. Data-handling was performed using theMicroCal Origin software (version 4.10), and the denaturationtemperature, Td (also called the melting temperature, Tm) is defined asthe temperature at the apex of the peak in the thermogram.

The results of DSC for Hafnia alvei phytase variants are summarized inthe Table 3 below.

TABLE 3 Comparative Thermostability of Hafnia alvei Phytases Td 1st ScanVariant (° C.) A150C/L259C 62.4 Q162N/R96N 63.8L16V/I310L/I313L/M319L/M354L 64.3 V87I/L103A/L112I/A113T/I114V 67.4E66C/L370C 67.2 H363R 68.3 Q162N/G186S 67.5 E54C/A101C 68.0V130L/M137L/V146I/I201V/M260L/I266V 67.8 Wt 69 K45P 70.9 K139C/I201C70.8 E54C/A101C/K139C/I201C 72.6 D92Y/E100W/A217G/H363R 75.1Q9S/N78Q/A89A/D92Y/H115M/A132/K139C/Q162/Q181L/ 75.5I201C/A217G/K234V/P348R Y48H/D92Y/E100W/K139C/T152I/I201C/A217G/K234V/77.7 N247D/H363R D92Y/E100W/K139C/I201C/A217G/N247D/H363R 78.0Y48H/D92Y/E100W/K139C/I201C/A217G/K234V/N247D/ 78.9 R289W/H363RE54C/D92Y/A101C/K139C/I201C/A217G/H363R 79.8E54C/D92Y/A101C/K139C/I201C/A217G/K234V/N247D/ 80.0 Q256D/H363RY48H/E54C/D92Y/A101C/K139C/T152A/I201C/A217G/ 80.2K234V/N247D/R289W/H363R Y48H/D92Y/E100W/K139C/I201C/A217G/K234V/N247D/81.7 Q256D/H363R Y48H/D92Y/T98S/E100W/K139C/T152A/I201C/A217G/ 82.7K234V/N247W/Q256/H363R Y48H/D92Y/E100W/K139C/T152A/I201C/A217G/K234V/83.1 N247W/Q256D/H363R D92Y/E100W/K139C/I201C/A217G/K234V/N247W/Q256D/84.2 H363R S1QGPS/K26H/E54C/A101C/K139C/Q162N/G186S/I201C/ 71.9K207Q/G346S

Example 5 Temperature Profile

The temperature profile (phytase activity as a function of temperature)was determined for the Hafnia alvei phytase and variants in thetemperature range of 20-90° C. essentially as described above(“Determination of phytase activity”). However, the enzymatic reactions(100 microliter phytase-containing enzyme solution+100 microliterssubstrate) were performed in PCR tubes instead of microtiter plates.After a 15 minute reaction period at desired temperature the tubes werecooled to 20° C. for 20 seconds and 150 microliters of each reactionmixture was transferred to a microtiter plate. 75 microliter stopreagent was added and the absorbance at 405 nm was measured in amicrotiter plate spectrophotometer. The results are summarized in Table4 below. The numbers given for each temperature are relative activity(in %) normalized to the value at optimum.

TABLE 4 Relative temperature profiles Temperature (° C.) Phytase variant20 30 40 50 60 65 70 75 80 85 90 *wt 18 29 50 75 100 94 93 24 12 7 5*K131Q 21 34 53 77 94 99 100 26 16 10 6 Q162N/D138N 17 28 44 66 88 10095 26 14 9 7 I294L 18 30 46 64 90 100 90 26 14 11 11 G72A 17 29 45 67 83100 78 26 13 11 8 H143C/I201C 10 22 37 62 85 100 91 26 5 5 4 Q162R 18 3049 73 91 100 95 26 15 11 9 I49L 17 29 45 65 90 100 94 26 13 9 11*E66C/L370C 15 27 43 66 100 88 99 27 12 6 5 T369S 18 28 47 64 92 100 9527 15 11 9 I201V 16 27 41 59 78 100 82 27 13 11 8 K148R 19 35 51 71 9897 100 27 11 7 6 A163K 14 27 38 63 79 100 93 27 12 9 6 S1QS 15 26 40 6287 93 100 27 9 4 2 F8M 15 27 38 67 96 100 91 27 10 5 5 F8Y 16 27 44 6996 100 93 27 10 6 6 T242S 18 30 48 66 91 100 93 27 12 11 10 K176R 17 3343 66 100 96 96 28 8 5 3 S403N 14 32 42 67 84 100 96 28 14 10 8 S1P 1727 44 60 87 100 92 28 13 11 8 E41Q 16 28 44 63 87 100 99 28 13 9 9*K131L 18 30 50 72 85 95 100 29 15 8 4 *K207R 18 30 48 70 88 93 100 2915 8 5 *K207Q 23 37 58 81 92 97 100 29 18 10 7 G346S 16 28 47 65 95 10092 29 10 6 4 T308A 9 17 33 56 80 99 100 29 12 9 6 S396K 19 31 44 69 86100 87 30 14 12 10 L401N 14 33 43 68 86 100 87 30 13 10 7 I201G 16 28 4362 81 100 87 30 14 11 8 P348R 18 30 43 62 81 100 91 31 14 11 8 E100W 1829 40 61 79 100 85 31 13 10 7 K187E 16 28 39 66 90 100 92 31 10 −6 4N239R 19 30 48 66 89 100 96 32 12 11 10 A304V 15 24 42 58 86 100 98 3214 11 8 S396D 16 28 41 60 81 100 87 32 13 10 8 T152G 16 28 42 66 85 10095 32 14 10 9 K12R 18 29 42 64 82 100 98 33 14 12 8 I303L 17 27 40 62 86100 88 33 13 11 10 Q162N 16 27 42 62 89 90 100 34 10 7 3 S192A 17 26 4359 88 100 94 35 13 9 9 T369D 18 29 43 66 86 100 90 35 14 12 11V130L/M137L/V146I/I201V/ 13 25 43 67 89 98 100 35 14 10 6 M260L/I266VQ109G 17 33 44 68 94 100 94 36 11 6 N239K 19 31 47 70 92 100 94 38 14 1211 K234V 17 29 42 63 79 100 94 39 15 11 8 K234E 17 27 43 61 86 100 92 4015 11 7 H363R 18 28 42 66 90 100 90 41 11 6 5 S261A 13 24 39 62 83 98100 41 14 10 7 A217G 14 32 41 65 84 100 90 43 15 11 7 E54C/A101C/K207Q21 33 48 68 89 100 95 55 13 10 6 K45P 19 29 43 68 86 100 93 59 18 11 10E54C/A101C 16 27 42 64 85 100 88 64 9 7 4 D33C/E54C/A101C/Y179C 16 31 4567 94 100 82 74 9 6 3 E54C/A101C/K139C/I201C/ 18 30 41 62 85 80 100 8436 9 10 K207Q D92Y/E100W/A217G/H363R 6 13 27 50 89 85 100 84 32 10 5E54C/A101C/K139C/I201C 15 28 40 59 86 78 100 86 47 8 9 K139C/I201C 18 2537 56 91 91 100 87 15 7 4 S1QGPS/K26H/E54C/A101C/ 26 37 49 67 80 100 9290 18 13 10 K139C/Q162N/G186S/I201C/ K207Q/G346S Y48H/E54C/D92Y/A101C/10 20 39 63 85 81 100 92 58 11 6 K139C/T152A/I201C/ K207Q/V208T/A217G/K234V/N247D/R289W/ H363R T35A/Y48H/E54C/P75N/ 5 9 28 50 77 80 100 93 135 1 K76N/D77Q/N78T/D92Y/ A101C/K139C/T152A/ I201C/A217G/K234V/N247D/R289W/H363R Y48H/E54C/D92Y/A101C/ 5 13 26 53 76 83 100 95 75 11 4K139C/T152A/I201C/V208T/ A217G/K234V/N247D/ R289W/H363RE54C/D92Y/A101C/K139C/ 10 15 28 52 81 93 100 96 83 10 1I201C/A217G/H363R E54C/D92Y/A101C/K139C/ 9 16 33 58 81 84 100 96 79 18 6I201C/A217G/K234V/N247D/ Q256D/H363R D92Y/E100W/K139C/I201C/ 6 13 27 4985 84 100 98 71 13 6 A217G/K234V/N247D/H363R P75N/K76N/D77Q/N78T/ 6 1530 51 81 82 100 98 76 20 5 D92Y/E100W/K139C/I201C/ A217G/K234V/N247D/Q256D/H363R Y48H/D92Y/E100W/K139C/ 8 11 25 44 78 88 100 99 68 10 2I201C/A217G/K234V/ N247D/R289W/H363R Q9S/N78Q/A89A/D92Y/ 8 16 32 54 8586 100 100 81 14 6 H115M/A132V/K139C/ Q162R/Q181L/I201C/A217G/K234V/P348R Y48H/D92Y/E100W/K139C/ 3 12 24 46 77 89 85 100 75 14 1I201C/A217G/K234V/N247D/ Q256D/H363R Y48H/D92Y/E100W/K139C/ 4 12 24 4576 92 87 100 66 14 4 T152I/I201C/A217G/K234V/ N247D/H363RD92Y/E100W/K139C/I201C/ 9 16 22 47 70 81 89 100 78 24 6A217G/K234V/N247W/ Q256D/H363R D92Y/E100W/K139C/D173N/ 6 16 37 52 81 8988 100 37 9 3 P175S/I201C/A217G/K234V/ N247D/Q256D/H363RY48H/D92Y/E100W/K139C/ 9 15 22 47 76 91 90 100 68 18 6I201C/V208T/A217G/K234V/ N247D/Q256D/H363R Y48H/E54C/D92Y/A101C/ 9 16 2657 78 89 90 100 80 21 7 K139C/I201C/A217G/K234V/ N247D/R289W/H363RY48H/D92Y/E100W/K139C/ 9 12 19 46 68 76 85 100 77 29 4I201C/A217G/K234V/ N247W/Q256D/H363R Y48H/D92Y/E100W/K139C/ 8 12 20 4666 78 80 100 81 33 4 T152A/I201C/A217G/K234V/ N247W/Q256D/H363RY48H/E54C/D92Y/A101C/ 4 8 21 44 80 91 88 100 81 17 3K139C/T152A/I201C/A217G/ K234V/N247D/R289W/ H363R Y48H/E54C/D92Y/A101C/7 13 22 48 76 87 84 100 82 24 6 K139C/I201C/A217G/K234V/N247W/R289W/H363R Y48H/E54C/D92Y/A101C/ 5 11 20 46 77 89 79 100 81 27 4K139C/T152A/I201C/A217G/ K234V/N247W/R289W/ H363R

TABLE 5 Heat stability at 75° C., activity relative to maximum activityRelative Mutation activity wt 24 K131Q 26 Q162N/D138N 26 I294L 26 G72A26 H143C/I201C 26 Q162R 26 I49L 26 E66C/L370C 27 T369S 27 I201V 27 K148R27 A163K 27 S1QS 27 F8M 27 F8Y 27 T242S 27 K176R 28 S403N 28 S1P 28 E41Q28 K131L 29 K207R 29 K207Q 29 G346S 29 T308A 29 S396K 30 L401N 30 I201G30 P348R 31 E100W 31 K187E 31 N239R 32 A304V 32 S396D 32 T152G 32 K12R33 I303L 33 Q162N 34 S192A 35 T369D 35V130L/M137L/V146I/I201V/M260L/I266V 35 Q109G 36 N239K 38 K234V 39 K234E40 H363R 41 S261A 41 A217G 43 E54C/A101C/K207Q 55 K45P 59 E54C/A101C 64D33C/E54C/A101C/Y179C 74 E54C/A101C/K139C/I201C/K207Q 84D92Y/E100W/A217G/H363R 84 E54C/A101C/K139C/I201C 86 K139C/I201C 87S1QGPS/K26H/E54C/A101C/K139C/Q162N/G186S/I201C/ 90 K207Q/G346SY48H/E54C/D92Y/A101C/K139C/T152A/I201C/K207Q/ 92 V208T/A217G/K234V/N247DR289W/H363R T35A/Y48H/E54C/P75N/K76N/D77Q/N78T/D92Y/A101C/ 93K139C/T152A/I201C/A217G/K234V/N247D/R289W/H363RY48H/E54C/D92Y/A101C/K139C/T152A/I201C/V208T/ 95A217G/K234V/N247D/R289/H363R E54C/D92Y/A101C/K139C/I201C/A217G/H363R 96E54C/D92Y/A101C/K139C/I201C/A217G/K234V/N247D/ 96 Q256D/H363RD92Y/E100W/K139C/I201C/A217G/K234V/N247D/H363R 98P75N/K76N/D77Q/N78T/D92Y/E100W/K139C/I201C/ 98A217G/K234V/N247D/Q256D/H363R Y48H/D92Y/E100W/K139C/I201C/A217G/K234V/99 N247D/R289W/H363R Q9S/N78Q/A89A/D92Y/H115M/A132V/K139C/Q162R/ 100Q181L/I201C/A217G/K234V/P348R Y48H/D92Y/E100W/K139C/I201C/A217G/K234V/100 N247D/Q256D/H363R Y48H/D92Y/E100W/K139C/T152I/I201C/A217G/ 100K234V/N247D/H363R D92Y/E100W/K139C/I201C/A217G/K234V/N247W/ 100Q256D/H363R D92Y/E100W/K139C/D173N/P175S/I201C/A217G/ 100K234V/N247D/Q256D/H363R Y48H/D92Y/E100W/K139C/I201C/V208T/A217G/ 100K234V/N247D/Q256D/H363R Y48H/E54C/D92Y/A101C/K139C/I201C/A217G/ 100K234V/N247D/R289W/H363R Y48H/D92Y/E100W/K139C/I201C/A217G/K234V/ 100N247W/Q256D/H363R Y48H/D92Y/E100W/K139C/T152A/I201C/A217G/ 100K234V/N247W/Q256D/H363R Y48H/E54C/D92Y/A101C/K139C/T152A/I201C/ 100A217G/K234V/N247D/R289W/H363R Y48H/E54C/D92Y/A101C/K139C/I201C/A217G/100 K234V/N247W/R289W/H363R Y48H/E54C/D92Y/A101C/K139C/T152A/I201C/ 100A217G/K234V/N247W/R289W/H363R

Example 6 pH Profile

The pH profile was determined at 37° C. in the pH range of 2.0 to 7.5(in 0.5 pH-unit steps) as described above in the section “Determinationof phytase activity”, except that a buffer cocktail (50 mM glycine, 50mM acetic acid and 50 mM Bis-Tris was used instead of the 0.25 M sodiumacetate pH 5.5 buffer. The results are summarized in table 1 below. Thevalues given for each pH in the range of 2.0-7.5 are the relativeactivity in % normalized to the value at optimum.

TABLE 6 Relative pH profiles at 37° C. pH Mutation 2 2.5 3 3.5 4 4.5 55.5 6 6.5 7 7.5 K26P 72 89 100 96 81 67 55 37 21 8 1 3 K26Q 40 69 91 10098 95 84 59 35 12 −1 1 K26R 48 66 85 100 99 98 87 64 40 14 2 −1D92Y/E100W/ 63 78 94 100 95 76 46 20 7 1 −1 9 A217G/H363R Q9S/N78Q/A89A/50 68 90 100 99 79 42 17 3 0 0 5 D92Y/H115M/ A132V/K139C/ Q162R/Q181L/I201C/A217G/ K234V/P348R T35C/L172C 39 65 87 100 99 94 73 51 30 10 1 1D92Y/E100W/ 63 78 94 100 95 76 46 20 7 1 −1 9 A217G/H363R Q9S/N78Q/A89A/50 68 90 100 99 79 42 17 3 0 0 5 D92Y/H115M/ A132V/K139C/ Q162R/Q181L/I201C/A217G/ K234V/P348R H143C/I201C 49 71 89 100 93 90 77 55 33 12 2 0Y48H/E54C/D92Y/ 42 65 89 100 98 85 53 21 7 1 0 −1 A101C/K139C/I201C/A217G/ K234V/N247D/ R289W/H363R Y48H/E54C/D92Y/ 47 62 89 100 97 8552 18 6 0 0 0 A101C/K139C/ T152A/I201C/ A217G/K234V/ N247W/R289W/ H363RT35A/Y48H/E54C 41 74 88 100 96 78 49 17 5 0 −1 −2 P75N/K76N/D77Q/N78T/D92Y/A101C/ K139C/T152A/ I201C/A217G/ K234V/N247D/ R289W/H363RY48H/E54C/D92Y/ 39 70 87 100 94 82 52 21 7 2 1 −1 A101C/K139C/T152A/I201C/ A217G/K234V/ N247D/R289W/ H363R E54C/D92Y/A101C/ 40 65 87100 99 88 57 23 7 1 0 −1 K139C/I201C/ A217G/H363R Y48H/E54C/D92Y/ 40 7084 100 98 90 65 29 11 3 1 −1 A101C/K139C/ T152A/I201C/ V208T/A217G/K234V/N247D/ R289W/H363R K131P 50 64 83 95 100 99 95 83 65 36 5 3 K131Q51 67 82 98 100 99 95 80 69 39 7 −2 K207R 38 51 71 85 100 97 95 81 50 273 3 D33C/Y179C 40 53 72 86 100 92 83 65 38 18 2 5 G325C/T358C 40 54 7893 100 100 90 68 41 16 0 −3 H228C/H363C 36 53 75 92 100 97 88 67 38 16 2−1 A150C/L259C 36 57 78 95 100 97 89 64 42 16 3 −2 D92Y/E100W/ 58 73 9099 100 84 49 20 6 1 −1 3 K139C/I201C/ A217G/N247D/ H363R D92Y/E100W/ 5873 90 99 100 84 49 20 6 1 −1 3 K139C/I201C/ A217G/K234V/ N247D/H363RT308A 39 58 79 99 100 94 85 76 55 36 4 −3 E54C/A101C 42 55 77 98 100 9085 63 40 15 2 1 Y48H/D92Y/E100W/ 54 68 84 98 100 93 54 20 5 0 0 −6K139C/T152I/I201C/ A217G/K234V/ N247D/H363R K131Q 51 67 82 98 100 99 9580 69 39 7 −2 I49L 40 62 83 97 100 94 80 58 37 14 2 −1 Y48H/E54C/D92Y/42 62 87 96 100 81 50 22 6 0 0 −1 A101C/K139C/ I201C/A217G/ K234V/N247W/R289W/H363R Q162N 40 66 75 96 100 93 91 73 47 22 6 1 Y48H/D92Y/E100W/ 4669 86 95 100 79 55 23 9 2 0 0 K139C/201C/ A217G/K234V/ N247D/R289W/H363R Y48H/D92Y/E100W/ 41 59 75 94 100 95 66 24 6 1 0 −1 K139C/T152A/I201C/A217G/ K234V/N247W/ Q256D/H363R E66C/L370C 33 58 80 94 100 100 8766 41 14 2 0 E41Q 44 62 78 93 100 97 86 68 41 17 3 0 Q109G 37 57 77 92100 100 96 73 48 23 4 0 A163K 47 63 75 92 100 99 89 67 41 13 3 0Y48H/D92Y/E100W/ 50 55 72 91 100 91 57 19 4 −1 −1 −1 K139C/I201C/A217G/K234V/ N247D/Q256D/ H363R A304V 53 60 82 91 100 95 88 68 45 19 3 3E54C/D92Y/A101C/ 35 48 68 90 100 94 64 22 6 0 −1 −1 K139C/I201C/A217G/K234V/ N247D/Q256D/ H363R P75N/K76N/D77Q/ 42 55 71 88 100 95 62 236 0 0 0 N78T/D92Y/E100W/ K139C/I201C/ A217G/K234V/ N247D/Q256D/ H363RD92Y/E100W/ 37 51 65 89 100 97 69 20 2 −3 −5 −4 K139C/I201C/A217G/K234V/ N247W/Q256D/ H363R Y48H/D92Y/T98S/ 39 50 69 89 100 99 73 288 0 −1 −1 E100W/K139C/ T152A/I201C/ A217G/K234V/ N247W/Q256D/ H363RK234E 44 53 75 88 100 99 95 71 47 19 3 1 D92Y/E100W/ 44 54 70 87 100 9362 22 5 1 0 −1 K139C/D173N/ P175S/I201C/ A217G/K234V/ N247D/Q256D/ H363RK207R 38 51 71 85 100 97 95 81 50 27 3 3 D33C/E54C/A101C/ 37 59 74 84100 88 82 65 41 21 5 2 Y179C Q162N/D138N 39 58 81 95 100 100 91 71 47 214 3 Q162R 38 58 78 88 100 100 96 72 48 22 3 0 N285D 45 61 81 96 100 10097 71 39 18 2 7 E66C/L370C 33 58 80 94 100 100 87 66 41 14 2 0 T369S 4656 79 92 100 100 94 72 47 22 3 −1 S192A 46 58 81 90 100 100 93 62 42 152 0 Y48H/E54C/D92Y/ 36 63 83 96 100 100 85 61 36 14 2 −1 A101C/K139C/T152A/I201C/ K207Q/V208T/ A217G/K234V/ N247D/R289W/ H363R wild type 4157 77 93 99 100 94 75 48 20 3 0 K131L 45 59 75 93 98 100 96 84 71 44 13−1 D33C/P178C 37 61 77 90 94 100 88 66 43 15 1 1 F63C/L368C 29 55 78 9298 100 83 65 38 14 2 −1 S1QS 45 56 80 86 99 100 83 63 45 14 3 0 I303L 4059 75 89 98 100 91 73 45 18 2 0 F8Y 44 59 78 93 98 100 88 64 37 14 3 −4I201V 36 53 73 91 98 100 88 68 40 13 −1 −2 Y48H/D92Y/E100W/ 46 60 76 8898 100 74 35 10 1 0 1 K139C/I201C/ V208T/A217G/ K234V/N247D/ Q256D/H363RK176R 38 58 76 90 97 100 94 72 45 18 0 −4 S1P 46 56 80 90 97 100 84 6440 15 2 −2 T242S 38 58 73 85 97 100 97 73 48 22 2 −1 S396D 41 54 76 8797 100 93 71 47 19 4 0 F8M 48 57 71 90 96 100 82 70 40 14 5 1 N239R 3246 69 93 96 100 98 74 50 24 1 2 N239K 38 52 70 91 96 100 95 73 48 20 3 0S396K 37 51 72 87 96 100 96 73 46 21 3 −1 K139C/I201C 34 48 69 84 96 10094 72 45 17 1 −2 K148R 35 57 70 90 95 100 92 75 47 15 1 −1 P348R 38 5473 91 95 100 98 72 48 25 3 1 I201G 36 52 73 93 95 100 89 68 41 14 −1 −5V130L/M137L/ 32 53 69 88 95 100 89 71 52 26 8 2 V146I/I201V/M260L/ I266VT152G 30 50 67 84 94 100 95 73 47 19 2 0 L401N 30 50 63 87 94 100 100 7250 23 2 1 I294L 37 53 78 83 93 100 94 73 46 20 2 −1 G346S 34 55 71 86 93100 90 69 43 18 2 −5 H363R 29 50 67 87 92 100 96 77 49 22 2 0 T369D 3554 68 82 92 100 90 72 51 18 3 0 G72A 35 47 67 85 90 100 80 61 37 14 −1−2 E54C/A101C/ 37 49 75 87 90 100 89 66 50 23 6 0 K139C/I201C S261A 3046 69 83 90 100 84 67 43 19 −1 −3 K187E 34 57 72 88 89 100 92 70 44 21 1−1 E54C/A101C/ 28 48 65 79 89 100 98 85 71 47 18 1 K139C/I201C/ K207QA217G 39 54 73 85 88 100 85 59 39 14 −1 −1 K207L 41 52 69 84 92 98 10092 69 41 11 1 K207Q 38 49 69 83 95 95 100 92 65 45 10 5 S403N 33 51 7285 99 99 100 77 49 22 1 −1 K45P 34 60 72 90 91 98 100 75 48 23 2 0 E100W29 44 69 78 93 98 100 84 57 28 3 0 S1QGPS/K26H/ 38 54 69 83 92 98 100 8864 29 2 −1 E54C/A101C/ K139C/Q162N/G186S/ I201C/K207Q/G346S K234V 35 5465 94 93 96 100 74 44 26 3 1 E54C/A101C/K207Q 34 52 70 85 96 96 100 9276 47 14 1 K207Q 38 49 69 83 95 95 100 92 65 45 10 5

Example 7 Steam Stability Method 1

Residual activity of phytase molecules after steam treatment wasevaluated using the following assay:

20 microliters of each purified enzyme sample is dispensed into a singlewell of a Corning® 96 Well (1×8 Stripwell™) plate (Corning, Lowell,Mass., USA) and subsequently evaporated to dryness in a vacuumcentrifuge (Genevac EZ-1 Plus, Genevac Ltd, Suffolk, UK). The steamincubation is performed in a closed styropor container with the innerdimensions 27×18×20 cm. The samples, in open strips, are placedapproximately 10 cm above the bottom of the container on a metal rack,in order not to be in contact with the water.

One liter of boiling water is poured into the container, the lid isclosed and the temperature of the produced steam monitored using athermometer mounted in the lid of the container. The incubation proceedsfor 60 seconds from the moment the water is poured into the container.During this period the temperature increases to about 85° C. Immediatelyafter the incubation the samples are cooled down on ice, re-suspendedand evaluated with respect to phytase activity using the colorimetricp-nitrophenyl phosphate (pNPP) assay (Sigma, Broendby, DK). Each enzymesample is compared to a similar sample that had not been steam treatedin order to calculate residual activity.

The results are presented in Tables 7 and 8 below.

TABLE 7 Steam Stability determined by method 1 Residual Activity Variant[%] Experiment 1 Wt 12 E66C/L370C 23 D33C/E54C/A101C/Y179C 22 Experiment2 Wt 14 G346S 25 Q109G 22 H143C/I201C 16 Experiment 3 Wt 10 Q162N(performed twice) 31; 35 Experiment 4 Wt 20E54C/D92Y/A101C/K139C/I201C/A217G/H363R 88Q9S/N78Q/A89A/D92Y/H115M/A132V/K139C/ 62Q162R/Q181L/I201C/A217G/K234V/P348RD92Y/E100W/K139C/I201C/A217G/N247D/H363R 51D92Y/E100W/K139C/I201C/A217G/N247D/Q256D/ 54 H363RY48H/D92Y/E100W/K139C/T152I/I201C/A217G/ 45 K234V/N247D/H363RY48H/D92Y/T98S/E100W/K139C/T152A/I201C/ 86 A217G/K234V/N247W/Q256D/H363RE54C/D92Y/A101C/K139C/I201C/A217G/K234V/ 91 N247D/Q256D/H363RY48H/D92Y/E100W/K139C/T152A/I201C/A217G/ 88 K234V/N247W/Q256D/H363RY48H/E54C/D92Y/A101C/K139C/T152A/I201C/ 90 A217G/K234V/N247D/R289W/H363RY48H/E54C/D92Y/A101C/K139C/T152A/I201C/ 95V208T/A217G/K234V/N247D/R289W/H363RT35A/Y48H/E54C/P75N/K76N/D77Q/N78T/D92Y/ 81A101C/K139C/T152A/I201C/A217G/K234V/ N247D/R289W/H363RY48H/E54C/D92Y/A101C/K139C/T152A/I201C/ 95K207Q/V208T/A217G/K234V/N247D/R289W/H363R Experiment 5 Wt 25.9D92Y/E100W/A217G/H363R   39.5 D92Y/E100W/K139C/I201C/A217G/N247D/H363R  57.5 Q9S/N78Q/A89A/D92Y/H115M/A132V/K139C/   68.5Q162R/Q181L/I201C/A217G/K234V/P348R

Method 2

In these experiments a modified set-up was used whereby the steam isprovided from a steam generator and led into the box. The samples placedon a plate are inserted into the box through a drawer when thetemperature has reached. Upon the insertion of the samples thetemperature drops 4° C. Incubation is performed for 30 seconds while thetemperature remains approximately constant at 90° C. Thereafter theplate is quickly removed from the box and the samples placed on ice. Thesamples are analyzed as in method 1.

TABLE 8 Steam Stability determined by method 2 Residual Activity Variant[%] Experiment 1 Wt 15 V130L/M137L/V146I/I201V/M260L/I266V 27 Experiment2 Wt 6 S1QGPS/K26H/E54C/A101C/K139C/Q162N/ 29 G186S/I201C/K207Q/G346SExperiment 3 Wt 4 S261A 12 T308A  9 Experiment 4 wt 8 L401N 14 S403N  9T308A 11 Experiment 5 wt 4 E54C/A101C  9 Experiment 6 wt 6 T152G 10Experiment 7 wt 5 S1P 10 F8M 14 Experiment 8 wt 3 K139C/I201C 13Experiment 9 wt 5 S1QS  9 A217G  9

Example 8 Glycation Residual Activity Inactivation by Glycation

The effect of glycation was investigated by incubation of purifiedphytase variants with glucose. For this 0.5 mg/ml enzyme in 0.1 M HEPESpH 7.5 was mixed with 1 M glucose and incubated at 50° C. for 5 hr. Thephosphatase activity was measured before and after incubation and theresults indicated below in Table 9.

TABLE 9 Modification of Glycation. Mutation Residual activity Wt 18%K26Q 55% K26R 47%

The results shown above indicate the wild type enzyme is stronglyinhibited by glycation (18% residual activity). Variants at position K26are clearly much improved in this respect being less affected.

Example 9 Pelleting Stability Tests Measurements of Pelleting Stability

Approximately 50 g enzyme granulate was pre-mixed with 10 kg feed for 10minutes in a small horizontal mixer. This premix was mixed with 90 kgfeed for 10 minutes in a larger horizontal mixer. From the mixer thefeed was led to the conditioner (a cascade mixer with steam injection)at a rate of approximately 300 kg/hour. The conditioner heated up thefeed to 95° C. (measured at the outlet) by injecting steam. Theresidence time in the conditioner was 30 seconds. From the conditionerthe feed was led to a Simon Heesen press equipped with 3.0×35 mmhorizontal die and pressed to pellets with a length of around 15 mm.After the press the pellets were placed in an air cooler and cooled for15 minutes.

Feed Formulation:

74.0% Grind corn

5.0% soy oil

20.7% Toasted soy grits

0.3% Solivit Mikro 106 premix of minerals and vitamins

12% water content

Test 1

A powder consisting of:

1.5 kg fibrous cellulose, Arbocel BC200

0.75 kg carbohydrate binder, Avedex W80

11.552 kg finely ground sodium sulphate

is granulated in a Lodige mixer FM 50 with a granulation liquidconsisting of:

0.75 kg carbohydrate binder, Avedex W80

2.49 kg Phytase Hafnia wt concentrate

0.45 kg water

The granulation is performed in a manner as described in U.S. Pat. No.4,106,991, Example 1. The obtained granulate is dried in a fluid bed toa water content below 1% and sifted to obtain a product with theparticle range 250 micro-m to 850 micro-m. Finally, the product iscoated with 10% palm oil and 22% calcium carbonate in a manner asdescribed in U.S. Pat. No. 4,106,991, Example 22.

Test 2

A powder consisting of:

1.6 kg fibrous cellulose, Arbocel BC200

0.80 kg carbohydrate binder, Avedex W80

12.027 kg finely ground sodium sulphate

is granulated in a Lödige mixer FM 50 with a granulation liquidconsisting of:

0.80 kg carbohydrate binder, Avedex W80

3.5 kg Y48H/D92Y/E100W/K139C/T152C/I201C/A217G/K234V/N247D/H363R variantconcentrate

0.02 kg water

The granulation, drying and sifting is performed as above. Finally, theproduct is coated with 9% palm oil and 22% calcium carbonate in a manneras above.

Test 3

A powder consisting of:

1.6 kg fibrous cellulose, Arbocel BC200

0.80 kg carbohydrate binder, Avedex W80

12.013 kg finely ground sodium sulphate

is granulated in a Lödige mixer FM 50 with a granulation liquidconsisting of:

0.80 kg carbohydrate binder, Avedex W80

3.2 kg phytase variant 2 concentrate

0.30 kg water

The granulation, drying and sifting is performed as above. Finally, theproduct is coated with 9.8% palm oil and 22% calcium carbonate in amanner as above.

Test 4

A powder consisting of:

1.6 kg fibrous cellulose, Arbocel BC200

0.80 kg carbohydrate binder, Avedex W80

12.225 kg finely ground sodium sulphate

is granulated in a Lödige mixer FM 50 with a granulation liquidconsisting of:

0.80 kg carbohydrate binder, Avedex W80

3.50 kg E54C/D92Y/A101C/K139C/I201C/A217G/H363R variant concentrate

The granulation, drying and sifting is performed as above. Finally, theproduct is coated with 9.0% palm oil and 22% calcium carbonate in amanner as above.

Test 5

A powder consisting of:

1.6 kg fibrous cellulose, Arbocel BC200

0.80 kg carbohydrate binder, Avedex W80

12.225 kg finely ground sodium sulphate

is granulated in a Lödige mixer FM 50 with a granulation liquidconsisting of:

0.80 kg carbohydrate binder, Avedex W80

3.50 kg Y48H/D92Y/E100W/K139C/I201C/A217G/K234V/N247D/Q256D/H363Rvariant concentrate

The granulation, drying and sifting is performed as above. Finally, theproduct is coated with 9.0% palm oil and 22% calcium carbonate in amanner as above.

Test 6

A powder consisting of:

1.6 kg fibrous cellulose, Arbocel BC200

0.80 kg carbohydrate binder, Avedex W80

12.435 kg finely ground sodium sulphate

is granulated in a Lödige mixer FM 50 with a granulation liquidconsisting of:

0.80 kg carbohydrate binder, Avedex W80

3.50 kg Y48H/D92Y/E100W/K139C/T152A/I201C/A217G/K234V/N247W/Q256D/H363Rvariant concentrate

0.30 kg water

The granulation, drying and sifting is performed as above. Finally, theproduct is coated with 9.7% palm oil and 22% calcium carbonate in amanner as above.

Test 7

A powder consisting of:

1.6 kg fibrous cellulose, Arbocel BC200

0.80 kg carbohydrate binder, Avedex W80

12.193 kg finely ground sodium sulphate

is granulated in a Lödige mixer FM 50 with a granulation liquidconsisting of:

0.64 kg carbohydrate binder, Avedex W80

3.50 kg phytase variant 5 concentrate

0.16 kg water

The granulation, drying and sifting is performed as above. Finally, theproduct is coated with 8.3% palm oil and 22% calcium carbonate in amanner as above.

The samples produced in Test 1 to Test 7 were tested in a pelletingtrial at 95° C., outlet of the conditioner. The phytase content wasmeasured using analytical method EB-SM 0559.02 version 01 (availablefrom Novozymes upon request) prior to pelletizing and in the feedpellets after pelletizing. The following residual activities of thephytase were found:

TABLE 10 Pelleting Stability Residual phytase activity Test [%] Variant1 13 Wt 2 26 Y48H/D92Y/E100W/K139C/T152I/I201C/A217G/ K234V/N247D/H363R3 20 D92Y E100W K139C I201C A217G K234V N247D H363R 4 30E54C/D92Y/A101C/K139C/I201C/A217G/H363R 5 26Y48H/D92Y/E100W/K139C/I201C/A217G/K234V/ N247D/Q256D/H363R 6 28Y48H/D92Y/E100W/K139C/T152A/I201C/A217G/ K234V/N247W/Q256D/H363R 7 43Y48H/E54C/D92Y/A101C/K139C/T152A/I201C/ A217G/K234V/N247D/R289W/H363R

The conclusion is that the variants have improved the pelletingstability compared to the reference Test 1.

Example 10 Performance in Animal Feed in an In Vitro Model

The performance in animal feed of a number of phytase variants of theinvention are compared in an in vitro model to the performance of areference protein such as SEQ ID NO:2. The in vitro model simulatesgastro-intestinal conditions in a monogastric animal and correlates wellwith results obtained in animal trials in vivo. The version used in thisexample simulates the crop and stomach of a broiler. The comparison isperformed as follows:

Phytase activity in the variant sample is determined as described inExample 1 under “Determination of phytase activity”.

Feed pellets from a broiler feeding trial—and with maize, soybean mealand soybean oil as main constituents—are pre-incubated at 40° C. and pH4.6 for 5 minutes followed by the addition of suitable dosages of thephytases (identical dosages are used for all phytases to be tested toallow comparison), for example between 125 to 1000 phytase units FYT/kgfeed, or buffer in the control samples. After 5 minutes of incubation,pepsin (3000 U/g feed) in an HCl-solution is added and in this way pH isreduced to 3. The samples are then incubated at 40° C. for another 5minutes.

The reactions are stopped and phytic acid and inositol-phosphatesextracted by addition of HCl to a final concentration of 0.5 M andincubation at 40° C. for 2 hours, followed by one freeze-thaw cycle and1 hour incubation at 40° C.

Phytic acid and inositol-phosphates are separated by high performanceion chromatography as described by Chen et al., 2003, Journal ofChromatography A 1018: 41-52 and quantified as described by Skoglund etal., 1997, J. Agric. Food Chem. 45: 431-436.

Degradation of phytate is then calculated as the difference ininositol-6-phosphate bound phosphorous (IP6-P) between phytase-treatedand non-treated samples. The relative performance of the variant iscalculated as the percentage of phytate degradation by the wild typephytase.

The relative degradation of the phytase variants (Table 11) show thatthe variants are all capable of degrading inositol-6-phosphate in the invitro system applied. Certain candidates performed better than the wildtype (e.g., variant: D92Y/E100W/A217G/H363R, variant:Y48H/D92Y/E100W/K139C/I201C/A217G/K234V/N247D/Q256D/H363R, variant:Y48H/D92Y/E100W/K139C/T152C/I201C/A217G/K234V/N247D/H363R) whereasothers were not as efficient in vitro as the wild type (e.g., variant:K207Q).

TABLE 11 In vitro degradation of IP6-P from a soybean/maize based diet.Phytate degradation of the variant is calculated as the percentage ofphytate degradation by the wild type phytase. Phytate degradation of thevariant as percentage of phytate degradation by the wild type Phytasedosage (two numbers represent data from Phytase variant (FYT/kg feed)two different trials) D92Y/E100W/A217G/H363R 125 181  As above 250 199 D92Y/E100W/K139C/I201G/A217G/K234V/N247D/H363R 125  74; 101 As above 250 71; 137 As above 500 72 As above 1000 76Y48H/D92Y/E100W/K139C/I201C/A217G/K234V/N247D/Q256D/H363R 125 252; 543As above 250 219; 347 As above 500 184; 215Y48H/D92Y/E100W/K139C/T152I/I201C/A217G/K234V/N247D/H363R 125 237; 297As above 250 160; 246 As above 500 148; 197 K207Q 250 57 E54C/A101C 250119  G346S 250 79 Q162N 250 180  S1QS 255  68* S1P 277  70* F8M 246  74*F8Y 229  71* K139C/I201C 250 105 S1QGPS/K26H/E54C/A101C/K139C/Q162N/G186S/I201C/K207Q/G346S 250 90 *Forthese data the wt was tested at 250 FYT/kg

Example 11 Performance in an In Vivo Pig Trial

Comparative evaluation of the effects of graded amounts of two Hafniaalveii phytase variants on the faecal digestibility and excretion ofphosphorus and calcium in growing pigs.

Sixty four Large White×Landrace pigs having an initial body weight of43.55±4.35 kg were used.

The animals were housed in floor-pen cages in an environmentallycontrolled room. Each pen had a plastic-coated welded wire floor and wasequipped with two water nipples and four stainless-stee individualizedfeeders. Room temperature was 21-22° C. and humidity percentage was 50%.

The pigs were fed a basal diet formulated to provide phosphorus (P)exclusively from vegetable origin during an adaptive period of 14 days.After that period they were allocated into 16 equal groups of 4 animalseach.

They were fed for 12 days the basal diet or this diet supplemented with1000, 2000 U/kg and 4000 U/kg of Hafnia alveii wild type phytase, with500, 1000 and 2000 U/kg of theY48H/D92Y/E100W/K139C/I201C/A217G/K234V/N247D/Q256D/H363R variant orwith 500, 1000 and 2000 U/kg of theY48H/D92Y/E100W/K139C/T152C/I201C/A217G/K234V/N247D/H363R variant.

An indigestible tracer (chromium oxide) was added at a concentration of0.4% to all the diets allowing calculation of the digestibility of P andcalcium (Ca). The feed was distributed ad libitum in mash form, underpen feed consumption control, and the animals had free access todrinking water. The digestibility of Ca was not corrected for Ca intakewith the drinking water.

Faecal P, Ca and Cr concentrations were measured at the 12^(th) day ofthe second period. Faeces were sampled individually, in approximatelythe same amount at the same time of the day, during the last 3 dayspreceding that date. Thus, for each dietary treatment and for eachcriterion a total of 12 individual determinations have been performed.All minerals were determined according to standard Association ofOfficial Analytical Chemists (1990) methods using a Vista-MPX ICP-OESspectrometer. The apparent digestibility (% of the intake) of theminerals was calculated for the mentioned 3 day period.

The mean P faecal concentration of the enzyme supplemented animals wasvery significantly lower than that observed for the animals ingestingthe control diet (a).

The P digestibility was dose depend and highly significantly improvedwith the five phytases in all supplemented groups (b). The highest Pdigestibility was observed in the 4000 U/kg Hafnia alvei wild typesupplemented diet and in the JHP113 group at 2000 U/kg.

The faecal excretion of P was significantly reduced in all the phytasesupplemented animals and for all the tested inclusion levels (c).

The apparent absorbed P was higher than the 2.25 g/kg recommended forthe growing pigs in the 4000 U/kg Hafnia alvei wild type group and veryclose to it with JHP113 and “C. braakii” wild type at 2000 U/kg and 4000U/kg respectively (d).

The P equivalences, considered as supplemental P digested comparativelyto the non-supplemented control, were highly significantly greater tothe control in all five phytases supplemented diets (e).

The Ca digestibility was improved and the Ca faecal excretion reducedwith all tested enzymes and at all inclusion levels (f).

The maximum of efficiency on these parameters was observed with Hafniaalvei wild type at the inclusion level of 4000 U/kg, whereas theY48H/D92Y/E100W/K139C/I201C/A217G/K234V/N247D/Q256D/H363R variantperformed the best when comparing the efficacy at the 1000 U/kg and 2000U/kg supplementations.

The results are presented in the following Table 12

TABLE 12 Residual levels of parameters for digestibility Dose (U/kg) 0500 1000 2000 (a) Phosphorus fecal concentration (mg/g DM) Wt 14.3 11.5Y48H/D92Y/E100W/K139C/I201C/A217G/ 13.7 12.6 11.5K234V/N247D/Q256D/H363R Y48H/D92Y/E100W/K139C/T152I/I201C/ 14.5 13.911.0 A217G/K234V/N247D/H363R Control 18.3 (b) Phosphorus apparent fecaldigestibility (%) Wt 47.3 50.1 Y48H/D92Y/E100W/K139C/I201C/A217G/ 44.155.3 58.0 K234V/N247D/Q256D/H363R Y48H/D92Y/E100W/K139C/T152I/I201C/43.1 48.6 51.4 A217G/K234V/N247D/H363R Control 27.9 (c) Phosphorusexcretion (mg/g DM) Wt 2.06 1.93 Y48H/D92Y/E100W/K139C/I201C/A217G/ 2.141.74 1.61 K234V/N247D/Q256D/H363R Y48H/D92Y/E100W/K139C/T152I/I201C/2.16 1.97 1.81 A217G/K234V/N247D/H363R Control 2.80 (d) Phosphorusabsorption (mg/g) Wt 1.85 1.94 Y48H/D92Y/E100W/K139C/I201C/A217G/ 1.692.16 2.22 K234V/N247D/Q256D/H363R Y48H/D92Y/E100W/K139C/T152I/I201C/1.63 1.86 1.91 A217G/K234V/N247D/H363R Control 1.09 (e) Phosphorusequvalences (mg/g) Wt 0.77 0.86 Y48H/D92Y/E100W/K139C/I201C/A217G/ 0.611.07 1.14 K234V/N247D/Q256D/H363R Y48H/D92Y/E100W/K139C/T152I/I201C/0.56 0.78 0.83 A217G/K234V/N247D/H363R Control 0.00 (f) Calcium apparentdigestibility (%) Wt 58.9 55.2 Y48H/D92Y/E100W/K139C/I201C/A217G/ 54.163.1 63.5 K234V/N247D/Q256D/H363R Y48H/D92Y/E100W/K139C/T152I/I201C/53.2 55.3 57.9 A217G/K234V/N247D/H363R Control 51.0

1. A phytase which has at least 76% identity to amino acid residues1-413 of SEQ ID NO:2 and which comprises at least one modification in atleast one position selected from the following: 139, 1, 4, 5, 6, 7, 8,9, 10, 12, 16, 18, 25, 26, 27, 28, 29, 30, 31, 32, 33, 35, 36, 37, 38,39, 40, 41, 45, 48, 49, 54, 55, 59, 63, 64, 66, 68, 69, 70, 71, 72, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 89, 91, 92, 93, 95, 96, 97, 98, 100,101, 103, 108, 109, 110, 111, 112, 113, 115, 116, 117, 118, 119, 120,121, 122, 123, 124, 125, 126, 128, 130, 131, 132, 133, 134, 136, 137,138, 140, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 158, 159, 160, 161, 162, 163, 168, 172, 173, 175, 176, 177,178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 189, 190, 192, 193,194, 195, 196, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,209, 211, 215, 217, 219, 221, 224, 227, 228, 230, 233, 234, 235, 236,238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 251, 256,258, 259, 260, 261, 266, 268, 270, 279, 284, 285, 286, 287, 288, 289,290, 292, 293, 294, 295, 296, 297, 298, 299, 301, 303, 304, 308, 310,312, 313, 314, 316, 318, 319, 320, 322, 324, 325, 326, 331, 335, 343,344, 345, 346, 347, 348, 354, 355, 356, 358, 360, 362, 363, 364, 365,366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 378, 382, 383,384, 385, 386, 387, 388, 389, 390, 391, 394, 395, 396, 397, 400, 401,403, 404, 406, 408, 409, 411, 412, and 413, wherein the positionscorrespond to the positions of the phytase with the amino acids 1-413 ofSEQ ID NO:2, with the proviso that the phytase is not the Hafnia alveiphytase with the amino acids 1-413 of SEQ ID NO:2. 2-42. (canceled)